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DETERMINATION OF BIOLOGICAL ACTIVITIES:

A LABORATORY MANUAL

Maria do Carmo Barreto

Nelson Simões

Editors

Protocols from the Workshop “Searching for bioactive compounds with potential applications in Biotechnology and Biomedicine”, Biopharmac Project, Universidade dos Açores, Ponta Delgada, 5-7 May 2010.

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EDITORSMaria do Carmo Barreto Nelson Simões

EDITION AND FUNDINGProject BIOPHARMAC (MAC/1/C104), co-financed at 85% by the European Union (FEDER), MAC 2007-2013.

CITATION GUIDEWhen quoting the bookBarreto, M.C. & Simões, N. (eds.) 2012. Determination of Biological Activities. A Laboratory Manual. Universidade dos Açores, Ponta Delgada.

When quoting a chapterMoujir, L.L., Cabral, C, Barreto, M.C. 2012. Determination of antimicrobial activities. In: Barreto, M.C. & Simões, N. (eds.), Determination of Biological Activities. A Laboratory Manual, pp. 5-25. Universidade dos Açores, Ponta Delgada.

ExECUçãO GRáFICAEGA - Empresa Gráfica Açoreana

DEpóSITO LEGAL341230/12

ISBN978-972-8612-82-5

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pREFACE

BIOPHARMAC stands for “Development of the biotechnological and pharmaceutical industry based on knowledge, research and development (R&D) in the framework of Macaronesian biodiversity”. This project, co-financed at 85% by the European Union (FEDER), MAC 2007-2013, involves as partners the Cabildo Insular de Tenerife (Project Coordinator), Fundación Canaria del Instituto Canario de Investigación del Cáncer, Universidade dos Açores, Universidade da Madeira and Universidad de La Laguna.

The main objectives of BIOPHARMAC are: (i) to promote high quality R&D in biotechnology and pharmaceutics in Macaronesia; (ii) to contribute to the transfer of knowledge and technology to the biotechnological and pharmaceutical industries; (iii) to create a transnational network of cooperation involving universities, R&D centers, investors and biotechnological and pharmaceutical industries in Macaronesia; and (iv) to contribute to the creation of new technology-based biotechnology and pharmaceutical industries in this region.

In this context, the Workshop “Searching for bioactive compounds with potential applications in Biotechnology and Biomedicine” took place in the Azores University in May 2010, and brought together specialists from Portugal and Spain. From the 5th to the 7th of May 2010, a group of students had the opportunity to learn state of the art techniques of determination of biological activities in the lab.

This manual presents the protocols from the workshop, with some additions which were considered useful by the editors and authors. Each chapter begins by an introduction which will allow the manual to be useful not only to the students who participated in the course but to any researcher that wants to start doing bioactivity determinations. Maria do Carmo Barreto Nelson Simões

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EDITORS / AUThORSMaria do Carmo Barreto - DCTD, Universidade dos Açores / CIRN (Centro de Investigação em Recursos Naturais), Portugal Nelson Simões - DB, Universidade dos Açores / CIRN / IBB-CBA (Instituto de Biotecnologia e Bioengenharia - Centro de Biotecnologia dos Açores), Portugal

AUThORSCarla Cabral - DB, Universidade dos Açores / CIRN / IBB-CBA, PortugalDuarte Toubarro - CIRN / IBB-CBA, PortugalElisabete Rego - DCTD, Universidade dos Açores / CIRN, PortugalJoana Serôdeo Medeiros - DCTD, Universidade dos Açores / CIRN, PortugalJorge Humberto Leitão - Biological Sciences Research Group (BSRG) Institute for Biotechnology and Bioengineering (IBB) /Instituto Superior Técnico, Universidade Técnica de Lisboa, PortugalJosé Silvino Rosa - DB, Universidade dos Açores / CIBIO-Açores (Centro de Investigação em Biodiversidade e Recursos Genéticos), PortugalLaila Moujir Moujir - Facultad de Farmacia de la Universidad de La Laguna, SpainLuísa Oliveira - DB, Universidade dos Açores / CIRN / IBB-CBA, PortugalMiguel Arruda - DCTD, Universidade dos Açores, PortugalNuno Rainha - DCTD, Universidade dos Açores, PortugalTânia Teixeira - CIRN, PortugalVera Gouveia - DCTD, Universidade dos Açores / CIRN, Portugal

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CONTENTS

1. Natural products as a source of new drugs and compounds with biotechnological application


2. Determination of antimicrobial activitiesLaila Moujir Moujir, Carla Cabral, Maria do Carmo Barreto

2.1. Methods of antimicrobial susceptibility testing2.1.1. Disk diffusion in agar platesProtocol 1: Assay for antibacterial and antifungal activity using the disk diffusion method Protocol 2: Screening of microorganisms for new antibiotics 2.1.2. Determination of antimicrobial activity by the broth dilution methodProtocol 3: Determination of MIC and MBC by the broth serial dilu-tion method in microplates2.2. Evaluation of mechanisms of action2.2.1. Changes in membrane integrityProtocol 4: BacLight fluorescent Syto 9 staining 2.2.2. Interaction with topoisomeraseProtocol 5: Inhibition of DNA gyrase

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131717192123

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3. Identification of novel antimicrobials using a live-animal infection modelJorge Humberto Leitão, Nelson Simões

Protocol 6: Cultivation and maintenance of Caenorhabditis elegansProtocol 7: Synchronization of Caenorhabditis elegans wormsProtocol 8: Nematode killing assaysProtocol 8b: Infection of C. elegans and determination of antimi-crobial action

4. Determination of cytotoxicity against tumour cell linesLaila Moujir Moujir, Vera Gouveia, Duarte Toubarro, Maria do Carmo Barreto

4.1. Growth and maintenance of adherent cell linesProtocol 9: Adherent cell line growth and maintenance 4.2. Cytotoxicity assaysProtocol 10: Cytotoxicity assays with adherent cell lines4.3. Determination of mechanisms of cell death by fluorescenceProtocol 11: Seeding cells for fluorescence microscopyProtocol 12: Fluorescence staining protocols

5. Cell-free assaysMaria do Carmo Barreto, Miguel Arruda, Elisabete Rego, Vera Gouveia, Joana S. Medeiros, Nuno Rainha

5.1. Interaction with enzymes5.1.1. Inhibition of acetylcholinesterase Protocol 13: Inhibition of acetylcholinesterase5.2. Antioxidant assaysProtocol 14: DPPH radical scavenging assayProtocol 15: Ferric chloride reduction assayProtocol 16: Folin-Ciocalteau total phenolics determination

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303133

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42434647535758

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66666872737678

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6. Testing insecticide activitiesJosé Silvino Rosa, Tânia Teixeira, Luísa Oliveira

Protocol 17: ovicidal activityProtocol 18: Contact toxicityProtocol 19: Antifeedant activity

AppENDIxESAppendix 1Maria do Carmo Barreto

A.1.1. McFarland Scale A.1.2. Correlation between CFU and Absorbance

Appendix 2Jorge Humberto Leitão

A.2.1. Nematode growth media A.2.2. Maintenance of C. elegans – freezing and thawing

Appendix 3Laila Moujir Moujir, Maria do Carmo Barreto

A.3.1 Cell line suppliersA.3.2. Media and materials for mammalian cell cultureA.3.3. Freezing and thawing cell culturesA.3.4. Counting cells in a haemocytometer

Appendix 4Maria do Carmo Barreto, Vera Gouveia, Duarte Toubarro

A.4. Coating growth surfaces with polylysine (for fluorescence or other microscopy observation of cells

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1. NATURAL pRODUCTS AS A SOURCE OF NEw DRUGS AND COMpOUNDS wITh

BIOTEChNOLOGICAL AppLICATION


The use of Nature for pharmacological and biotechnological applications is thousands of years old, very likely far older than the first written accounts dating from the Assyrians and the Egyptians, the Chinese and the Indians. After a long history of empirical use of medicinal preparations, mainly derived from terrestrial plants, the birth of modern investigation in Natural Products can be traced back to the 19th century, with the isolation of the first active principles, such as morphine and salicylic acid.

In the 20th century, until the 1980s, many studies were carried out to isolate and identify the structure of active compounds from natural sources, mainly terrestrial plants but also fungi and some marine invertebrates. In the last two decades of the century, there was some decline in the research of Natural Products by the pharmaceutical industry. This was mainly due to the development of virtual high throughput screening by computational methods, which extended the possibilities of pharmacore design to molecules which might not necessarily exist physically but which might be synthesised to bind to an enzyme or another drug target.

The results from using this approach were less promising than expected, and this led to a renewed interest in the search for active molecules from Nature, either as pharmacophores or as scaffolds do develop new molecules with the required characteristics. The great majority of compounds which passed from the initial

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research, into proof of concept, clinical phase studies and finally registration as approved drugs is either of natural origin or modified from molecules of natural origin. Natural product structures are chemically diverse, possess biochemical specificity and other properties due to millions of years of evolution (hence the term “evochemistry”), which make them favorable as lead structures for drug discovery.

The search for bioactive molecules in Nature relies on an interdisciplinary approach which involves at least Biologists, Natural Products Chemists, Molecular Biologists, Biochemists and specialists in Bioinformatics. Several approaches are possible to choose a possible source of pharmacophores, usually terrestrial plants, macroalgae or sessile marine invertebrates that synthesize active molecules as defense compounds. Studying plants traditionally used for their medicinal properties, or that are actively avoided by predators, for instance, may be a good strategy.

Whatever the approach, the investigation will sooner or later require some kind of bioassay (Fig. 1.1).

Fig. 1.1. Bioassay-guided fractionation as a tool to discover molecules with pharmacological potential.

A bioassay or biological activity assay is an experiment which assesses the activity of a given extract or compound against a biological target (Table 1.1). This assay can be, for example: (i) a chemical reaction indicative of a given effect in a living system (e.g., many antioxidant assays); (ii) interference with a subcellular system, such as enzymes, receptors and organelles; (iii) growth inhibition of microorganisms, like bacteria; (iii) cytotoxicity or growth arrest of cell cultures derived from human or other origin; (iv) toxicity against live invertebrates as whole organism models.

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Table 1.1 Examples of biological activity assays

Activity Target

Antioxidant DPPH radical, H2O2, other oxidant or pro-oxidant molecules

Interference with enzymes Acetylcholinesterase inhibition

Antibacterial activity Human, animal or plant pathogens, (e.g, Staphylococcus aureus, Escherichia coli)

In vitro cytotoxicity HeLa, MCF-7 or other tumour cell lines

Whole-organism toxicity Caenorhabditis elegans, Artemia salina, Pseudaletia unipuncta

If properly designed and carried out, a biological activity assay will give information not only on whether a given effect is to be expected in a living organism, but also on the relative magnitude and selectivity of that effect. This may be useful, for example, when a bioassay-guided fractionation is being carried out, in which the fractionation and purification of a given molecule is guided by the increase in bioactivity, corresponding to the enrichment of the active compound. When testing a high number of pure compounds of known structure, combining experimental with in silico (i.e., computational) assays may be an option, since it may help to narrow the number of molecules to be tested in the lab.

After purifying a bioactive compound, its mechanism of action is characterized. This requires further experiments, but it is also at this step that computational models are more useful. These models integrate biological and medical data from many sources and can make predictions and suggest hypothesis about the interaction between, for example, a molecule and a biological receptor. There are also models which help to predict the absorption, distribution, metabolism, excretion and toxicity of a given molecule.

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REFERENCES

Biggs, R.D. 2005. Recent Advances in the Study of Assyrian and Babylonian Medicine. Journal of Assyrian Academic Studies 19, 1-4

Butler, M.S., Buss, A.D. 2006. Natural products -The future scaffolds for novel antibiotics? Biochemical Pharmacology 71, 919– 929

Chin, Y-W., Balunas , M.J., Chai , H.B., Kinghorn, A. D. 2006. Drug Discovery From Natural Sources. The AAPS Journal 8, E239-E253

Cragg, G.M., Grothaus, P.G., Newman, D.J. 2009. Impact of Natural Products on Developing New Anti-Cancer Agents. Chemical Reviews 109, 3012–3043

Ekins, S., Mestres, J. Testa, B. 2007. In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling. British Journal of Pharmacology 152, 9–20

Koehn, F.E., Carter, G.T. 2005. The evolving role of natural products in drug discovery. Nature Reviews 4, 206-220

McCloud, T.G. 2010. High Throughput Extraction of Plant, Marine and Fungal Specimens for Preservation of Biologically Active Molecules. Molecules 15, 4526-4563

Müller, W.E.G., Schröder, H.C. , Wiens, M., Perovic-Ottstadt, S., Batel, R., Müller, I.M. 2004. Traditional and Modern Biomedical Prospecting: Part II—the Benefits Approaches for a Sustainable Exploitation of Biodiversity (Secondary Metabolites and Biomaterials from Sponges). Evidenced-based Complementary and Alternative Medicine 1, 133–144

Newman D.J. , Cragg G.M. 2007. Natural Products as Sources of New Drugs over the Last 25 Years. Journal of Natural Products 70, 461-477

Wilson, Z.E., Brimble, M.A. 2009. Molecules derived from the extremes of life. Natural Product Reports 26, 44–71

Zips, D., Thames, H.D., Baumann, M. 2005. New Anticancer Agents: In Vitro and In Vivo Evaluation. In Vivo 19, 1-8

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2. DETERMINATION OF ANTIMICROBIAL ACTIVITIES

Laila Moujir Moujir, Carla Cabral, Maria do Carmo Barreto

The increase in pathogenic bacteria resistant to most antibiotics has led to a renewed interest in the search for new antimicrobial agents, particularly those with novel mechanisms of action. After more than two decades (from the 1980’s) in which the pharmaceutical industry invested its efforts mainly in combinational chemistry, further investigation of antimicrobial molecules in natural sources, such as plants, algae, marine invertebrates and microorganisms, has gained a new emphasis.

These natural sources have yielded new and interesting compounds, which may be small molecules such as are often found in plants or algae, or antimicrobial peptides which are more common in invertebrates, fungi and bacteria. These molecules can either be used as new antibiotics or be the scaffolds to develop molecules with im-proved properties.

Whatever the strategy involved in the discovery of new products, sooner or later an experimental assessment of their antimicrobial activity will be required. Different methods can be used, including agar diffusion methods (paper disk or wells) and the broth dilution method.

2.1. Methods of antimicrobial susceptibility testing.

With the introduction of a variety of antimicrobials it became necessary to perform antimicrobial susceptibility tests as a routine. For this effect, the antimicrobial agent

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is allowed to diffuse out into the medium and interact in a plate or container freshly seeded with the test organisms. A variety of antimicrobial containing systems are used but the antimicrobial impregnated absorbent paper disk is by far the common-est type used. All techniques involve either diffusion of antimicrobial agent in agar or dilution of antibiotic in agar or broth.

2.1.1. Disk diffusion in agar plates

Müeller-Hinton agar is considered to be the best medium for routine susceptibility testing of nonfastidious bacteria and that meets the criteria of the Clinical and Labo-ratory Standards Institute (CLLSI, former NCCLS). In the agar diffusion method, activities are expressed as the diameter of growth inhibition (in mm) around the paper disk or well containing the compound. Although the size of the inhibition zone is proportional to its activity, the solubility of the compound in the agar must be considered. This method is useful for initial screenings of activity and to assess the spectrum of activity against different microorganisms (e.g., Gram positive and Gram negative bacteria, fungi).

The diffusion methods are those most often employed in research in spite of certain diffi culties, but they are models with a low credibility for samples that are diffi cult to diffuse in the media, because of the relationship between diffusion power and antimicrobial activity.

PROTOCOL 1: Assay for antibacterial and antifungal activity using the disk

diffusion method

Materials and reagents

Tubes with 4-5 mL of sterile tryptic soy broth for inoculum

Sterile saline solution

25-30 mL of Müeller-Hinton agar for each plate with a diameter of 100 mm

100 mm plates

Disk of 6 mm in diameter (Whatman fi lter paper no.1) and cork borer

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PROTOCOL 1: continued

DMSO (dimethylsulfoxide)

Ethanol

Micropipettes

Sterile Tips

Sterile cotton swabs

Tubes with 9 mL of buffer saline

McFarland standards

Methods

Preparation of extract/ pure compound stock solutions: weigh and dissolve in 1. the appropriate diluents (DMSO, ethanol) to yield the required concentration. Recommended concentrations are 20 mg/ mL for extracts and 5 mg/ mL for pure compounds.

Prepare dilutions from stock solutions to 1000, 500 and 100 μg/mL (pure com-2. pounds) and 10.000, 5000 and 1000 μg/mL (extracts) in DMSO or ethanol.

Add 10 μL of each dilution to a sterile paper disk and let dry at room tempera-3. ture, in sterile environment (e.g., in a laminar fl ow hood).

Inoculum preparation: At least three to fi ve well-isolated colonies of the same 4. morphological type are selected from an agar plate culture. The top of each colony is touched with a loop, and the growth is transferred into a tube contain-ing 4 to 5 mL of a suitable broth medium, such as tryptic soy broth. The broth culture is incubated at 35ºC until it achieves or exceeds the turbidity of the 0.5 McFarland standard (usually 2 to 6 hours). The turbidity of the actively grow-ing broth culture is adjusted with sterile saline or broth to obtain a turbidity optically comparable to that of the 0.5 McFarland standard. This results in a suspension containing approximately 1 to 2 x 108 CFU/mL for E.coli ATCC 25922.

Optimally, within 15 minutes after adjusting the turbidity of the inoculum 5.

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suspension, a sterile cotton swab is dipped into the adjusted suspension. The swab should be rotated several times and pressed fi rmly on the inside wall of the tube above the fl uid level. This will remove excess inoculum from the swab.

The dried surface of a Müeller-Hinton agar plate is inoculated by streaking 6. the swab over the entire sterile agar surface. This procedure is repeated by streaking two more times, rotating the plate approximately 60° each time to ensure an even distribution of inoculum. As a fi nal step, the rim of the agar is swabbed.

The predetermined battery of disks with the compound is dispensed onto 7. the surface of the inoculated agar plate. Each disk must be pressed down to ensure complete contact with the agar surface.

As an alternative, instead of incorporating the disks, perform wells (6 mm) 8. with a sterilized cork borer (or with the broad size of a glass Pasteur pipette, sterilized by immersing in alcohol and fl aming). Add 10 μL of each concen-tration of compound/extract to the wells, but dissolved in saline.

Incubate at optimum temperature for 24-48 h.9.

Fig. 2.1. Illustration of the agar diffusion method using paper disks. On the left, a result of this test is presented, with inhibition zones of different sizes. On the right, a schematic representation of an inhibition zone.

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Interpretation of results: The diameters of the zones of complete inhibition (as judged by the unaided eye) are measured, including the diameter of the disk/well, as seen on Figure 2.1.

pROTOCOL 2: Screening of microorganisms for new antibiotics

Many bacteria and fungi produce compounds which inhibit the growth of compet-ing microorganisms or even cause their death. Among these are broad-spectrum classical antibiotics, organic acids, lytic agents such as lysozyme, several types of protein exotoxins and bacteriocins with bactericidal action. Testing the anti-microbial action of bacteria requires separating the producing bacteria from the antimicrobial compounds, so that the only bacteria whose growth will be quanti-fi ed in the antimicrobial assays will be the target bacteria (“indicator strain”). Several strategies can be used to attain this end, which differ in the case of non spore-forming and spore-forming bacteria.

Materials and Reagents

Plates with nutrient agar (NA)

Tubes with 5 ml of soft NA (nutrient broth with 7 g of bacteriological agar/L)

Culture of indicator strain ( approximately 10 4 CFU / mL)

Chloroform

Inoculating loop

Micropipettes

Sterile Tips

Cellulose acetate membranes (0.45 μM, 85 mm diameter)

Methods 2.a. non spore-forming bacteria

1. Inoculate a nutrient agar plate or other suitable agar medium with the produc-

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ing bacterium. To do this remove 2-3 colonies from a fresh culture and place them into a small circle on the agar (Fig. 2.2).

Fig. 2.2. Testing of non spore-forming bacteria. A, producer bacteria are seeded and small circles and allowed to grow for 24-48h; B, after producer bacteria are killed by chloroform vapors, a layer of soft NA inoculated with target bacteria is poured over the underlying agar; C, after incubation, inhibition zones reveal the presence of antibacterial agent(s).

2. Incubate the plate for 24-48 hours at the adequate temperature.

3. Submit the plate to chloroform vapors for 30-60 minutes to kill the bacteria.

4. Aerate the boxes for 1-2 hours to remove traces of chloroform.

5. Fill a tube with 5 ml of soft NA (nutrient broth with 7 g of bacteriological agar/L).

6. Inoculate the tube with 100 µL of a culture of the indicator strain in exponential phase, with approximately 104 CFU / mL. Homogenize and pour over the Petri dish containing the dead producing bacteria.

7. Incubate the Petri dishes at the optimum temperature for the producer bacte-ria.

8. Note the presence of inhibition zones around the producing bacteria in positive cases (Fig. 2.2).


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2.B. spore-forming bacteria

1. Place a Petri dish with nutrient agar in an incubator at 37 º C for 1 hour, to remove humidity.

2. Place a cellulose acetate membrane with 0.45 μm pore size and 85 mm diameter on top of the sterile agar (Fig. 2.3).

Fig. 2.3. Testing of spore-forming bacteria. Producing bacteria are seeded on a 0.45 μm membrane which allows passage of compounds to underlying NA.After incuba-tion and membrane removal, a layer of soft NA inoculated with target bacteria is poured over the underlying agar; after incubation for growth of target bacteria, inhibi-tion zones reveal the presence of antibacterial agent(s).

3. Remove 2-3 colonies from a fresh culture of the producing bacterium and deposit them in a small circle on the membrane, avoiding the edge of the membrane (Fig. 2.3).

4. Incubate the plates overnight at the appropriate temperature.

5. Carefully remove the membrane.

6. Fill a tube with 5 ml of soft NA (nutrient broth with 7 g of bacteriological agar).

7. Inoculate the tube with 100 µL of a culture of the indicator strain in exponential phase, with approximately 104 CFU / mL. Homogenize and pour over the Petri dish.

8. Incubate the plates at the right temperature.

9. Note the presence of inhibition zones in positive cases (see Fig. 2.3 and 2.4).

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Fig. 2.4. Result of a test of antimicrobial activity produced by spore-forming bacteria.

2.1.2. Determination of antimicrobial activity by the broth dilution method

The broth dilution method is used to determine the minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC). The MIC is defined as the lowest con-centration of the compound which is able to inhibit the growth of bacteria, and the MBC as the lowest concentration which is able to cause the death of 99.9% of the bacterial population. These tests, if carried out on a larger scale, involve the preparation of a series of tubes containing culture media and an increasing concen-tration of test substance, and inoculated with the same amount of bacteria. After incubating at the appropriate temperature and time, the concentration of microor-ganism is quantified by the turbidity that appears in the tubes (e.g., by measuring absorbance at 600 nm or comparing with McFarland standard). A recount of viable microorganisms may also be carried out at the end of the incubation (Fig. 2.5).

These tests are time consuming but are more quantitative than agar diffusion meth-ods (e.g. they also allow the determination of EC50 and EC90 concentrations) and are a good choice when studying substances with low diffusion rates, or when testing activity against slow-growing bacteria or that require special culture media which change the diffusion rate.

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Fig. 2.5. An example of the broth macro dilution method to assess susceptibility of bacteria to a compound. Drawing by: Manuel Rodríguez López.

The scaling down of macro broth dilution methods to 96-well microplates (micro dilution) has the advantages of requiring less amount of compounds to test and of being much faster, since in each microplate a high number of concentrations can be tested simultaneously.

pROTOCOL 3: Determination of MIC and MBC by the broth serial dilution method in microplates

Equipment

Microplate reader ( e.g., Biorad Model 680).

Laminar fl ow hood (clean bench)

Incubator


Nutrient broth (NB)

Sterile 96 well microplates

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Test compound, fi lter-sterilized and diluted in nutrient broth (in a concen-

tration which is twice the highest concentration to be used)

Micropipettes

Multichannell micropipette

Sterile tips

Sterile 1.5 mL microtube (“eppendorf” tube)

Exponentially growing cultures of target bacteria in nutrient broth (0.5

McFarland Scale or 1 x 108 CFU/mL).

Diluted cultures of target bacteria in nutrient broth (1 x 10 5 CFU/mL).

50 mg/mL stock solutions of test compounds in DMSO, ethanol or other

solvent miscible with water

Solutions of test compounds in nutrient broth (diluted from stock solu-

tions), at 80 μg/mL (pure compounds) or 200 μg/mL (extracts).

Method

1. In each row of the microplate, a different substance can be tested against the target bacteria. However, it is better to use at least 3-4 rows per compound to al-low for experimental error.

2. Pipette 200 µL of sterile nutrient broth into all wells in column 1. In wells from columns 3 to 12, pipette 100 µL of sterile nutrient broth.

3. In column 2, put 200 µL of the test compound solution diluted in nutrient broth in each well (Fig. 2.6, red or green circles), twice the maximum concentration re-quired for the assay.

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Fig. 2.6. Preparation of the microplate to determine antibacterial activity by the se-rial dilution broth method – for two compounds, with four replicates each

4. With a multichannel micropipette calibrated to 100 µL, aspirate several times from column 2 to mix, and remove 100 µL to wells 3 (Fig. 2.7). Repeat until wells 11. The remaining 100 µL are rejected (this will create a serial dilution from wells 2 to11, where each well has half the concentration of the previous one, see Fig. 2.8

Fig. 2.7. Serial dilution of the two compounds from columns 2 to 11 (same two com-pounds, with four replicates each).

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5. Do not add compound to test in wells 12. Wells in column 1 and 12 will serve as controls (wells 1 as blank and 12 as untreated bacteria) .

6. In wells 2 to 12 add 100 µL of the 1 x 105 CFU/mL target bacteria culture.

Fig. 2.8. After serial dilution of the two compounds from columns 2 to 11, the mi-croplate has decreasing concentrations of each compound, where each column has ½ the concentration of the previous one.

7. Incubate the plates for 16-24 hours at the appropriate growth temperature, preferably with agitation.

8. In a microplate reader, measure the optical density at 550 - 600 nm.

9. Determine the MIC and MBC for each sample against the target bacteria.

10. The MIC value can be determined by the lowest concentration that complete-ly inhibits growth of the organism in the tubes or microdilution wells as detected by the unaided eye. It can also be expressed as the concentration that reduces op-tical density by 50% or 90% (MIC50 or MIC90), when compared with the optical density of untreated bacteria (column 12).

11. The MBC value is determined by sub culturing 100 μL of the samples in agar, and verifying which was the minimum concentration that produced no viable bacteria.

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Notes:

Several substances can be tested on the same plate, but only one species of

target bacterium, to avoid cross contamination.

When stock solutions of compounds to test are prepared in solvents such as

Dimethylsulfoxide (DMSO) or methanol, it is not necessary to fi lter-steril-ize the compound before dissolving it in NB, since this solvents at almost 100% concentration are effective in killing the bacteria.

2.2. Evaluation of mechanisms of action

Compounds with antimicrobial activity may be classifi ed according to their mecha-nisms of action. The fi rst and most basic classifi cation is related with the degree of reversibility of the antimicrobial effect. If the compounds kill the bacteria, e.g. by the production of hydroxyl radicals that induce dramatic and irreversible changes leading to cell death, they are classifi ed as bactericidal. Bacteriostatic compounds, on the con-trary, are those that inhibit growth and reproduction of bacteria without killing them, i.e, they have a potentially reversible effect, for example by interfering with the synthesis of bacterial DNA, protein, cell wall components or other aspects of cell metabolism.

2.2.1 Changes in membrane integrity

The destabilization of the bacterial membrane by foreign substances can cause loss of membrane integrity, permeability changes and functional changes which ulti-mately trigger cell death. For many bactericidal agents, the action is initiated by their interaction with the cell membrane.

Several methods may be used to detect changes in membrane integrity, such as measuring leakage of intracellular constituents to the extracellular medium, e.g., po-tassium, inorganic phosphate, amino acids, materials absorbing at 260 nm, nucleic acids and proteins.

Methods which use fl uorescent markers have the advantage of being very sensitive and precise. One of these methods used to detect damage in the bacterial cell mem-

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brane is BacLight SYTO 9. In this procedure, cells are treated with two DNA binding dyes, Propidium Iodide (PI) and SYTO 9. SYTO 9 diffuses through the intact cell membrane and binds to its DNA showing a green fluorescence, while PI is highly charged and only penetrates bacteria with damaged membranes, causing red fluores-cence upon binding to DNA. Therefore all cells will present green fluorescence, but only cells with damaged membranes will show red fluorescence. When using both dyes, intact cells will be green and cells with damaged membranes will be mostly red, since the simultaneous presence of the two dyes causes a reduction in the fluo-rescence of SYTO 9 (Figs. 2.9 and 2.10). This double staining method allows the effective separation between viable and dead cells in mixed populations.

Fig. 2.9. BacLight SYTO 9 double staining reveals morphology and changes in membrane integrity of Bacillus subtilis cells. A, control. B and C, treated for 1 hour with 15 μg/mL and 25 μg/mL zeylasterone, respectively. D, treated for 1 hour with dimethylzeylasterone. Photo: Laila Moujir Moujir.

A B

C D

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Fig. 2.10. Fluorescence images of Staphylococcus aureus dyed with BacLight SYTO 9. A, control, B, bacteria treated with clofoctol for 30 minutes. Photo: Laila Moujir Moujir.

PRotoCoL 4: BacLight fl uorescent Syto 9 staining

Equipment

Fluorescence microscope

Laminar fl ow hood

Centrifuge

Thermostatic horizontal shaking bath


Nutrient broth

Dimethylsulfoxide (DMSO)

Saline solution

Propidium iodide (PI)

SYTO 9

Clofoctol (or other standard compound known to damage bacterial cell

membrane), in DMSO

Compound solution(s) in DMSO

A B

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Culture(s) of target bacteria

Monod fl asks

Microtubes (eppendorf tubes)

Micropipettes

Sterile tips

Microscope slides and coverslips

Method

1. Start with a pre-inoculum, incubated under stirring at 37°C for 18 to 24 hours.

2. Transfer 200 µL of the pre-inoculum to a Monod fl ask containing 20 mL of nutrient broth (NB).

3. Incubate with agitation at 37°C until the culture enters exponential phase.

4. Transfer 5 mL of this culture to each of the fl asks (one for control, the others depending on the number of products or concentrations to test).

5. Add products to their respective fl asks and DMSO to control fl ask.

6. Incubate for 1 hour at 37°C with orbital shaking.

7. Draw 1 mL from each fl ask and centrifuge at 10,000 rpm for 10 minutes at 4°C.

8. Eliminate the supernatant and add 1 mL of saline solution to resuspend cells.

9. Incubate for 30 minutes.

10. Centrifuge at 10,000 rpm for 10 minutes at 4°C.

11. Eliminate the supernatant and resuspend once more in saline.

12. Centrifuge again under the same conditions.

13. Eliminate the supernatant.

14. Resuspend in saline (1 mL).

21


15. Preparation of propidium iodide and SYTO 9 mixture: Mix 5 µl of SYTO 9 and 5 µl of propidium iodide in the dark

16. Add 1.5 µL of the mixture to each microtube containing bacteria and homog-enize gently.

17. Observation on the fl uorescence microscope: transfer approximately 5 µL of each sample to the corresponding microscope slide and cover with a coverslip. For PI use G-2A fi lter (510-560 nm) and for SYTO 9, FITC fi lter (488 nm).

2.2.2. Interaction with topoisomerases

DNA gyrase is a type II topoisomerase which is present in all bacteria and intro-duces negative supercoiling in the bacterial DNA, helping to separate its strands. The DNA molecule is fi rst twisted, subsequently there is a cleavage in a region where both strands are in contact and fi nally the cleaved strands are reunited on the opposite side of the intact chain (Fig. 2.11).

Fig. 2.11. Schematic representation of the mechanism by which DNA gyrase causes the relaxation and supercoiling of bacterial DNA (adapted from Mandigan et al., 2004).

The inhibition of DNA gyrase interrupts the replication and repair of DNA, tran-scription, the separation of daughter strands during bacterial replication and also other cellular processes in which DNA is involved. Compounds like quinolones, synthetic molecules which inhibit gyrase and topoisomerase IV, another enzyme which uncoils DNA during replication, are excellent broad-spectrum antibiot-ics.

22

The effect of test compounds on DNA gyrase is assessed by agarose gel electropho-resis (Figs. 2.12 and 2.13)

Fig. 2.12. Gel electrophoresis of DNA gyrase inhibition detection kit (DNA Gyrase Assay Cat # 1003, TopoGen, 2000).

Fig. 2.13. Effect of celastrol on pBR322 supercoiling by DNA gyrase. A, control. B, control with DMSO. C and D, positive controls: 25 and 50 μg/mL ciprofloxacin. E, 50 μg/mL celastrol. F, 50 μg/mL pristimerin. Photo: Laila Moujir Moujir.

Analysis of Fig. 2.13 shows that celastrol (E) was able to inhibit DNA gyrase, in a manner similar to control ciproflaxin at 50 μg/mL (D), whilst pristimerin (F) pre-sented no effect at the same concentration.

23

PRotoCoL 5: Inhibition of DNA gyrase

The determination is carried out according to the indications of the manufacturer. In the present protocol, the DNA Gyrase Assay Kit 1, John Innes Enterprises, is used. Two Units (2 U ) of DNA gyrase are incubated with 0.5 μg pBR322 relaxed plasmid in a fi nal volume of 30 μL, in the presence of different concentrations of the compounds to test (see http://www.gencompare.com/john_innes_enterprises.htm, accessed 11/1/2012, for information on this kit).

5A. DNA Gyrase Assay

Materials

John Innes DNA gyrase kit (pBR322 relaxed plasmid, 5X gyrase buffer,

gyrase)

Milli –Q water

Method

1. Add 0.5 μL of the relaxed pBR322 (1 μg/mL) to each eppendorf tube.

2. Add 6 μL of 5X gyrase buffer to each eppendorf.

3. Add 15 μL of the test compounds, diluted in MiliQ water to twice the required fi nal concentrations (the fi nal volume in the assay is 30 μL). For example, to test a concentration of 50 μg/mL, prepare 50 μL of a 100 μg/mL solution in water.

4. Add 2 μL of gyrase (2 U).

5. Adjust the volume to 30 μL with distilled water.

6. For each experiment, prepare a control eppendorf with the same volume of the solvent used to dissolve the products, and also a positive control with ciprofl oxa-cin (a synthetic chemotherapeutic antibiotic of the fl uoroquinolone drug class).

7. Incubate the reaction mixture for 30 minutes at 37ºC. The result is analyzed by electrophoresis (part B).

24


5B. Electrophoresis protocol

Solutions

50X TAE buffer (Tris / acetate / EDTA)

TRIS Base 24,2 gGlacial Acetic acid 5,71 gNa2EDTA. 2H2O 3,72 gH2O until 100 mL

1X TAE buffer (Tris / acetate / EDTA)

50X TAE buffer 2 mLDistilled water 98 mL

Electrophoresis loading buffer

Bromophenol blue (Sigma) 0.025 gGlycerol (Sigma) 3 mLDistilled water until 10 mL

0.7% Agarose Gel

Low EEO D-1 Agarose /Pronadisa) 0.525 gTAE buffer 1X 75 mL

Make a concentrated (50x) stock solution of TAE by weighing out 242 g Tris base and dissolving in approximately 750 mL deionized water. Carefully add 57.1 mL glacial acetic acid and 37.2 g Na2EDTA.2H2O and adjust the solution to a fi nal volume of 1 L. This stock solution can be stored at room temperature. The pH

25


of this buffer is not adjusted and should be about 8.5. The working solution of 1x TAE buffer is made by simply diluting the stock solution by 50x in deionized water. Final solute concentrations are 40 mM Tris acetate and 1 mM EDTA.

Method

1. Boil 75 mL of 0.7% agarose (e.g., 30-60 seconds in a microwave oven) until it is dissolved and clear.

2. Allow it to cool until it is around 60ºC, pour in the electrophoresis tray and allow it to become solid (Fig. 2.14).

3. Add 1X TAE to the gel to keep it moist, and more 300 mL to the electrophore-sis system (the volume will depend on the size of the system you use).

Fig. 2.14. Preparation of the agarose gel for electrophoresis. Drawing: Manuel Rodríguez López.

4. Add 5 µl of bromophenol blue to each sample (Fig. 2.15).

5. Pour the samples in the wells and run the gel (90 V / cm).

26


Fig. 2.15. Preparation of the samples and electrophoresis. Drawing: Manuel Rodríguez López.

6. Reveal the gel in Ethidium Bromide for 10 minutes.

7. Wash the gel with water for 10 minutes and photograph.

27

REFERENCES

Andrews, J.M. 2001. Determination of minimum inhibitory concentrations. Journal of Anti-microbial Chemotherapy 48, Suppl. S1, 5-16

Barton, L.L.. 2005. Structural and Functional Relationships in Prokaryotes. New York, Springer, 820 p. ISBN 0-38-720708-2

Butler, M., Buss, A. 2006. Natural products — The future scaffolds for novel antibiotics? Biochemical Pharmacology 71, 919-929

Chatterji M., Unniraman S., Mahadevan S. and Nagaraja V. 2001. Effect of different classes of inhibitors on DNA gyrase from Mycobacterium smegmatis. Journal of Antimi-crobial Chemotherapy 48, 479-485

DNA Gyrase Assay Cat # 1003, TopoGen, 2000

Gradisar H., Pristovsek P., Plaper A., and Jerala R. 2007. Green Tea Catechins Inhibit Bac-terial DNA Gyrase by Interaction with Its ATP Binding Site. Journal of Medicinal Chemistry 50, 264-271

Galm U., Heller S., Shapiro S., Page M., Li S. and Heide L., 2004. Antimicrobial and DNA Gyrase-Inhibitory Activities of Novel Clorobiocin Derivatives Produced by Mu-tasynthesis. Antimicrobial Agents and Chemotherapy 48, 1307–1312

Kalemba D., Kunicka A. 2003. Antibacterial and Antifungal Properties of Essential Oils. Current Medicinal Chemistry 10, 813-829

Kim Y., Farrah S., Baney, R. H. 2007. Membrane damage of bacteria by silanols treatment. Electronic Journal of Biotechnology 10, 252-259

Kohanski, M.A., Dwyer, D.J., Hayete, B., Lawrence, C.A., Collins, J.J. 2007. A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics. Cell 130, 797–810.

LIVE/DEAD BacLight™ Bacterial Viability Kits Manual, Molecular Probes®

López M.R., de León L., Moujir L. 2011. Antibacterial Properties of Phenolic Triterpenoids against Staphylococcus epidermidis. Planta Medica 77, 726-729. DOI: 10.1055/s-0030-1250500

28

Moat, A. G., Foster, J. W., Spector, M. P., 2002. Microbial Physiology. 4th ed. New York, Wiley-Liss. 736 p. ISBN 0-471-39483-1

Mandigan, M.T., Martinko, J.M., Parker, J. 2004. Brock. Biología de los Microorganismos. Ed. Pearson Educación, S.A., Madrid, España

Motta, A.S., Cladera-Olivera, F., Brandelli, A. 2004. Screening for antimicrobial activity among bacteria isolated from the Amazon basin. Brazilian Journal of Microbiolo-gy 35, 307-310

National Committee for Clinical Laboratory Standards. 1997, Methods for dilution antimi-crobial susceptibility test for bacteria that grow aerobically 4th ed. NCCLS docu-ment M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa.

Prescott, L., Harley, J., Klein, D. 2002. Microbiología. Ed. McGraw Hill Interamericana de España S.A.U.

Queralt N., Araujo R., 2007. Analysis of the Survival of H. pylori Within a Laboratory-based Aquatic Model System Using Molecular and Classical Techniques. Microbial Eco-logy 54, 771–777

Rios, J.L., Recio, M.C., Villar, A. 1988. Screening methods for natural products with anti-microbial activity: a reviews of the literature. Journal of Etnopharmacology 23, 137-144

Spyzek, J., Novotná, J., Rezanka, T., Demain, A.R. 2010. Do we need new antibiotics? The search for new targets and new compounds. Journal of Industrial Microbiology and Biotechnology 37, 1241-1248

29

3. IDENTIFICATION OF NOVEL ANTIMICROBIALS usIng a lIve-anImal InfeCtIon model

Jorge Humberto Leitão, Nelson Simões

The increase in antibiotic-resistant pathogenic bacteria has led to the need for new therapeutic approaches to fight these infections. Using a live-animal infection model to screen novel antimicrobials is a useful tool to identify compounds which are ef-fective antimicrobials, while simultaneously testing their pharmacokinetic proper-ties and their toxicity to the host.

The nematode Caenorbhabditis elegans is a unique animal model which can be in-fected and killed by a large number of human pathogens, such as the Gram-negative bacteria Pseudomonas aeruginosa and Salmonella enterica, the Gram-positive bac-teria Enterococcus faecalis, Staphylococcus aureus and Bacillus cereus, and the fungus Cryptococcus neoformans.

There is a large overlap of bacterial virulence factors required for the appearance of pathogenicity in mammals and the ability to cause the death of C. elegans. It has also been shown that the main aspects of the innate immune system have been conserved between this nematode and mammals. These facts contribute to make C. elegans an ideal model to test antibiotics meant for human use.

The test is carried out by replacing the normal food source of C. elegans (in culture, usually Escherichia coli) with pathogenic bacteria, thus infecting the nematode. After the infection, the anti-microbial compound to be tested is dissolved in the nematode growth medium and the animal’s survival is monitored, and compared

30

with the appropriate controls. The test should be carried out in nematodes which are in the same developmental stage (the life cycle is shown in Fig. 3.1), therefore a synchronization protocol is also included below.

Fig. 3.1. Representative stages of the Caenorhabditis elegans life cycle.

pROTOCOL 6: Cultivation and maintenance of C. elegans

Equipments


Centrifuge

Incubator

Freezer

Materials

Petri dishes (plates)

Scalpel

Cryo tube

31


Reagents

PBS buffer

Glycerine (30%)

NGM I (1.5 g NaCl, 1.25 g Tryptone, 8.5 g agar, H 2O to 500 mL)

Organisms

E. coli OP50

Caenorhabditis elegans

Method

For laboratory use, Caenorhabditis elegans can be sustained on NGM I plates, covered with E. coli OP50, until three weeks at 20ºC.

1. Inoculate NGM I plates with 100 µL of a fresh overnight culture of E. coli and incubate plates at 37 ºC overnight. Overgrown plates can be stored at 4 ºC for several weeks.

2. During the assays, transfer nematodes every 2 days to fresh E. coli plates. For this, cut a piece of agar (covered with E. coli and C. elegans) with a sterile scalpel and put it onto the surface of a new E. coli plate.

pROTOCOL 7: Synchronization of Caenorhabditis elegans worms

Equipments


Centrifuge

Materials

Petri dishes (60 mm diameter) with C. elegans worms and eggs

32


Six-well plates (sterile)

2 mL tubes (sterile)

Reagents

H 2Obidist (sterile bidistilled water)

Sodium hypochlorite (12%)

NaOH (6N)

M9-buffer (3 g KH 2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Sterilize by autoclaving).

NGM I (Nematode Growth Medium, 1.5 g NaCl, 1.25 g Tryptone, 8.5 g

agar, additives, H2O to 500 mL). For details, see Appendix.

Method

To synchronize all C. elegans at the same development stage, use 60 mm Petri dishes with plenty of eggs (you’ll need 1 Petri dish for approximately 3 six-well plates). These eggs should be obtained 3-4 days after pouring a chunk of agar containing nematodes in a 60 mm Petri plate containing E. coli OP50.

1. For each Petri dish, prepare 3 empty 2 mL tubes. To an additional 2 mL tube, add 600 µL of H2Obidist plus 500 µL of sodium hypochlorite and 400 µL of 6 N NaOH and mix.

2. Rinse worms and eggs from the Petri dishes 4 times, with 1 mL of sterile H2Obidist, and disperse the suspension on three 2 mL tubes (disperse each rinsed mL dividing it by the three tubes).

3. Mix the suspensions in the 3 tubes, with an aliquot of 500 µL of the hypochlo-rite solution prepared in (1) per tube, by vortexing for approximately 7 minutes (maximum 10 minutes) until all worms have been dissolved.

4. Centrifuge for 1 minute (4 ºC, 3200 rpm or 800 g).

33


5. Discard the supernatant carefully and wash the pellet with 1 mL H2Obidist .

6. Centrifuge for 1 minute and discard the supernatant.

7. Resuspend the pellet of 1 tube with 100 - 200 µL of M9-buffer and use this re-suspension to re-suspend the pellets of the other tubes.

8. Pipette the solution on a NGM I Petri dish with E. coli and incubate at 20 ºC.

9. Inoculate 5 mL culture of the bacterial test strains on the same day. In the next 24h, inoculate in the six-well plates. After 24h, inoculate the nematode larvae on the six-well plates.

Note: egg preparation, inoculation of liquid cultures and of the six-well plates, and counting of surviving worms should be performed always at the same hour on the different days (more or less) so that the intervals do not exceed more than 24 hours.

pROTOCOL 8: Nematode killing assays

Equipments

Clean bench

Centrifuge

Materials

Petri dishes (60 mm diameter) with C. elegans worms

Cultures of pathogenic bacterial strains

E. coli OP50 culture

Six-well plates (sterile)

2 mL tubes (sterile)

34


Reagents

NGM II (Nematode Growth Medium II, 1.5 g NaCl, 1.75 g Bactopeptone,

8.5 g agar, H2O to 500 mL)

Methods

1. Adjust overnight cultures of the bacterial strains to a density of approximately 1.3 x 104 to 1.5 x104 CFU/mL.

2. Plate 100 µL of these bacterial suspensions on 6-well plates containing nema-tode growth medium NGM II, and place in an incubator at 37ºC for 24h.

3. After 24h a bacterial lawn should be formed. Inoculate the 6-well plates with approximately 25 hypochlorite-synchronized L4 larvae of C. elegans (see proto-col 7) per well and determine the actual number of worms.

4. Incubate plates at 20ºC and score for live worms after 1, 2, 3 and 5 days. Nematodes are considered dead when they fail to respond to touch.

5. Register the percentage of live worms and their morphological appearance after two days. Carry out all experiments at least fi ve times, and use E. coli OP50 as a negative control in the assays.

Note 1: according to Cardona et al., 2005, a strain is considered pathogenic to C. elegans if one of the following criteria is met: i) a sick appearance at day 2, in-cluding reduced locomotive capacity and the presence of a distended intestine, ii) percentage of live worms after 2 days ≤50%, and iii) total number of worms after 5 days ≤ 100. To differentiate mild from severe infections, the presence of one, two or three of these criteria can be scored as 1, 2 and 3 (PS 1, 2 and 3). While a strain is considered pathogenic when at least one criterion can be observed, a strain is described as non pathogenic when no symptoms of the disease are ob-served during the course of the experiment.

Note 2: in assays to identify the antimicrobial action of extracts of compounds, repeat the experiments in the presence of different concentrations of the com-pounds, using the appropriate controls.

35

pROTOCOL 8B: Infection of C. elegans and determination of antimicrobial action

Equipments


Centrifuge

Incubator

Materials

Petri dishes

Organisms and test compounds:

Caenorhabditis elegans (N2) and E. coli OP50

Pseudomonas aeruginosa and Bacillus sp.

Supernatants of bacterial growth

Compound or extracts in NGM

Antibiotic solutions for controls

Reagents:

Agar

Bacto-Peptone

LB Broth

NaCl

CaCl2

MgSO4

36

PROTOCOL 8B: continued

KH2PO4

Cholesterol

PBS or 0,8% NaCl as buffer

Distilled water

ngm (nematode growth medium) - preparation of agar plates:

1. NGM medium is prepared by autoclaving the following:

3 mg NaCl

2.5 mg Bacto-Peptone

17 mg Agar

975 mL distilled water

2. When the medium is cooled to 55 ºC, the following reagents are added in order, using sterile techniques:

1 mL cholesterol (5mg/mL in EtOH). Need not be sterilized.

1 mL 1 M CaCl 2

1 mL 1 M MgSO 4

25 ml KH 2PO4 (136 g of KH2PO4, add water to 900 mL. Adjust pH to 6.0 with concentrated KOH. Add water to make up to 1 liter. Autoclave.)

3. Mix and pour the medium into the Petri dishes.

37

PROTOCOL 8B: continued

Method

A. Infection with microorganisms

1. Prepare the bacterial cultures (E. coli, Bacillus sp., Pseudomonas aerugi-nosa or other microorganism) in an Erlenmeyer with LB broth medium and incubate overnight at 28ºC.

2. Prepare Petri-dishes with 5ml of NGM.

3. Inoculate NGM with culture of the bacterial strains and incubate overnight at 28ºC.

4. Transfer 10 nematodes in L4 stage to each NGM Petri-dish and keep over-night at room temperature.

5. Observe the nematodes at different times (check for mortality or any other modifi cation).

B. Antimicrobial compounds

1. Prepare three Petri-dishes with 5mL of NGM.

2. Prepare bacteria in liquid culture and incubate overnight at 28ºC.

3. Inoculate two NGM plates with E. coli and one with P. aeruginosa, and incubate overnight at 28ºC.

4. Apply the compound to test in one of the NGM Petri-dishes previously in-oculated with E. coli and on the the Petri dish with P. aeruginosa. Incubate for 1 hour. (E. coli without compound must be used as the control).

5. Transfer 10 nematodes in L4 stage and keep at RT.

6. Observe the nematodes at different times (check for mortality or any other modifi cation).

38

REFERENCES

Adonizio, A., Leal, S.M. Jr., Ausubel, F.M., Mathee, K. 2008. Attenuation of Pseudomonas aeruginosa virulence by medicinal plants in a Caenorhabditis elegans model sys-tem. Journal of Medical Microbiology 57, 809-13

Cardona, S.T., Wopperer, J., Eber, L., Valvano, M.A. 2005. Diverse pathogenicity of Bur-kholderia cepacia complex strains in the Caenorhabditis elegans host model. FEMS Microbiology Letters 250, 97-104

Mahajan-Miklos, S., Tan, M.W., Rahme, L.G., Ausubel, F.M. 1999. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model. Cell 96, 47-56

Mahajan-Mikos, S., Rahme L.G., Ausubel, F.M. 2000. Elucidating the molecular mecha-nisms of bacterial virulence using non-mammalian hosts. Molecular Microbiology 37, 981-988

Moy, T.L., Ball, A.R., Anklesaria, Z., Casadei, G., Lewis, K., Ausubel, F.M. 2006. Identifica-tion of novel antimicrobials using a live-animal infection model. Proceedings of the Natural Academy of Sciences of the USA 103, 10414-10419

Ramos, C.G., Sousa, S.A., Grilo, A.M., Eberl, L., Leitão, J.H. 2010. The Burkholderia ceno-cepacia K56-2 pleiotropic regulator Pbr, is required for stress resistance and viru-lence. Microbial Pathogenesis 48, 168-177

Sousa, S.A., Ramos, C.G., Almeida, F., Meirinhos-Soares, L., Wopperer, J., Schwager, S., Eberl, L., Leitão, J.H.. 2008. Burkholderia cenocepacia J2315 acyl carrier protein: A potential target for antimicrobials’ development? Microbial Pathogenesis 45, 331–336

Sousa, S.A., Ramos, C.G., Moreira, L.M., Leitão, J.H.. 2010. The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans. Microbiology 156, 896–908

Stiernagle, T. 1999. Maintenance of C. elegans. In: C. elegans. A practical approach. (I.A. Hope Ed.), Oxford University Press.

39

Tan, M.W., Rahme, L.G., Sternberg, J.A., Tompkins, R.G., Ausubel, F.M. 1999. Pseu-domonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeru-ginosa virulence factors. Proceedings of the Natural Academy of Sciences of the USA 96, 2408-2413.

“Caenorhabditis elegans WWW Server”, http://elegans.som.vcu.edu/ (accessed 16/02/2012)

“WormBase”, http://www.wormbase.org/ (accessed 16/02/2012)

41

4. DETERMINATION OF CYTOTOxICITY AGAINST TUMOUR CELL LINES

Laila Moujir Moujir, Vera Gouveia, Duarte Toubarro, Maria do Carmo Barreto

In vitro cytotoxicity assays provide a fundamental tool for the identification of com-pounds with potential antitumour action. Besides providing quantitative information on cytotoxic effects, these assays also allow the researchers to assess the selectivity of a given agent or extract, when carried out against a wide panel of representative cell lines.

Cell proliferation and viability can be assessed by different methods, such as (i) de-termination of cell integrity by measuring lactate dehydrogenase in the extracellular medium, (ii) measuring activity of mitochondrial succinate dehydrogenase by the MTT [3 - (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] method, or (iii) quantifying total proteins, like the SRB (sulforhodamine) method. The MTT and the SRB method are both extensively used, and apparently give the same proportional response to the amount of viable cells present, although each has its defenders. Ideally, the choice of method should take into consideration a profound knowledge of its advantages and disadvantages, such as interference with certain compounds which may affect the desired correlation between the signal detected and cell viability.

Experiments with in vitro cell lines are also useful to determine the mechanism of cell death. Compounds that cause necrosis in primary cell cultures have a non-acceptable cytotoxicity, whilst compounds that lead to the occurrence of apoptosis

42

are preferable for therapeutic use. The concentration of compounds and the kinetics of cell death may vary. However, the primary necrosis or cell lysis typically occurs very quickly after adding the product (2 hours or less). In turn, apoptosis proceeds in a slower and more organized way (4 - 48h). Unambiguous determination of the mechanism of cell death requires the use of several methods which concur in their result, such as microscopical analysis of cell morphology, fluorescence markers of apoptosis, determination of caspases and detection of DNA fractionation by electro-phoresis, among others.

4.1. Growth and maintenance of adherent cell lines

A great variety of culture cell lines, derived from tumour and non-tumour animal sources, can be obtained from tissue culture collections (e.g., the European Collec-tion of Cell Cultures, and the American Type Culture Collection). When screening extracts or compounds for antitumour activity, a panel of cell lines originated from different tumours should be used, since responses to a given chemical often vary with the cell line.

Some cell lines grow in suspension whilst others are anchorage-dependent (see be-low). The following protocols can be used for most adherent cell lines, although it is safer to confront these protocols with the information provided by the supplier for that particular cell line, considering that some lines may have specific require-ments.

Many cell lines, such as HeLa (human cervix epithelial carcinoma), are anchorage-dependent, i.e., they require attachment to a surface in order to survive and prolifer-ate. The surface of Petri dishes, culture flasks and other containers used for these cells is therefore treated in a way which encourages adherence of the cells, such as coating with polylysine, to name the most commonly used system.

There are several formulations of cell culture media which meet the requirements of most cell lines, including glucose, salts and amino acids. DMEM (Dulbecco’s Modified Eagle’s Medium) and RPMI (Roswell Park Memorial Institute) are two examples of essential media which are used in most cell culture labs. These media also contain phenol red, a pH indicator which helps to verify that the pH does not fall outside the range 7.2-7.4, ideal for most mammalian cells. Including bicarbonate in the media and maintaining an atmosphere of 5% CO2 atmosphere contributes to

43

maintain the pH at 7.4, together with HEPES buffer. Freshly prepared L-glutamine is also routinely added to the medium, since its half-life at 4ºC does not exceed 3 weeks. Fetal bovine serum is often included, since it provides several factors, some of which promote cell growth and adhesion. Antibiotics may also be added to the culture medium, although ideally it would be better to avoid them, since they may eventually alter the biochemistry of the cells.

Cell medium may have to be changed after 2-3 days if growth is very intense, since there may be nutrient depletion and accumulation of toxic metabolic products. Cells should be kept in exponential growth, before they become completely confl uent and cover the whole growing surface. Therefore, when they are almost confl uent, they should be passaged (i.e., removed from the fl ask, diluted and transferred to a new fl ask). This involves using proteolytic enzymes, usually trypsin, although some cell lines can be easily detached using EDTA only (EDTA chelates calcium, which is involved in the cadherin-mediated cell-cell adhesion).

Cell lines may be kept by cryopreservation, ideally in liquid N2, although when frozen at -80ºC they retain viability for at least 3 months. Cryopreserved cells are a precaution in case there is some contamination in the lab, and are also a way to ensure the length of time that the cells are grown in continuous culture, since con-tinuously subculturing a cell line may bring about genetic changes which alter its characteristics. Protocols for cryopreserving and thawing cells are included in Ap-pendix (Appendix 3, A.3.1).

pROTOCOL 9: Adherent cell line growth and maintenance

Equipment

Incubation chamber with CO 2 and humidity control (37ºC, 5% CO2 and 98% humidity)

Laminar fl ow hood (clean bench )

Thermostated water bath

Centrifuge

Inverted microscope

44



Petri dishes or T-fl asks (with modifi ed surface adequate for adherent cell

culture)

Pippeting aids

Sterile pipettes (reusable or disposable)

Sterile Pasteur pipettes (reusable or disposable)

Cell growth medium (DMEM with 5% FBS, antibiotic mixture and L-

glutamine, see Appendix)

EDTA

Trypsin / EDTA

15 mL sterile Falcon tubes in a tube rack

Container with diluted hypochlorite, to discard media (inside the clean

chamber)

Container with diluted hypochlorite for used materials (outside the cleam

chamber

Flame-sterilizing gas-burner

Methods

When cells are nearly confl uent (i.e., occupying most of the surface), they have to be subcultured, as mentioned above. For HeLa, this should be carried out every 2-3 days if DMEM with 5% FBS (D5%) is used. Using a lower FBS con-centration, e.g. 2% (D2%) will slow down growth and allow for larger lengths of time between passaging, e.g., during the weekend. Obviously all the work must be done in strict sterile conditions, so all manipulations involving the ma-nipulation of cells and media must be done in a clean chamber.

1. Before starting the work, place D5%, EDTA and Trypsin/EDTA in a shallow water bath at 37ºC. They should be placed in the laminar fl ow hood only when

45


starting the work, in order to be as close as possible to 37ºC.

2. Pipette 4.5 mL of D5% to one of the 15 mL sterile tubes.

3. Remove the cell culture from the incubator and place it inside the laminar fl ow hood. Discard the medium into the hypochlorite container.

4. Pour the content of 1-2 Pasteur pipettes with EDTA into the culture fl ask (the amount should be enough to cover the bottom of the fl ask and therefore to bathe all the cells.

5. Allow the EDTA to contact the cells for 1-2 minutes, gently swirling the culture fl ask to increase the contact. Discard the liquid onto the hypochlorite container.

6. With the Pasteur pipette, pour approximately the same amount of Trypsin / EDTA (T/E) into the fl ask. Place into the incubator at 37ºC for 3 minutes.

7. Remove the culture fl ask from the incubator. Back in the clean bench, detach the remaining cells by gently pipetting the T/E with a Pasteur pipette and fl ushing the bottom surface (cell detachment can be monitored at the microscope, although it is easily seen by tilting the fl ask inside the chamber and carefully observing the bottom – the aspect of cell layer and fl ask bottom is quite different).

8. Pour the cells suspended in T/E into the centrifuge tubes which already contain DMEM and centrifuge for 5-7 minutes at 200 g at room temperature.

9. Discard the supernatant and re-suspend the pellet in 1 mL D5%, gently pi-petting in and out of an 1 mL pipette (or with a Pasteur pipette). Add 6 mL more D5% to the tube and homogenize gently with the pipette.

10. Remove 1 mL of the homogenized cell suspension into the new culture fl ask and add D5% (the dilution and volumes should be adapted to the amount of start-ing material and also to the area of the fi nal culture vessel; e.g., 1 mL cells + 9 mL D5% for a 100 mm diameter Petri dish or 0.5 mL cells + 4.5 mL for a T25 fl ask).

Note: a higher concentration of cells will result in shorter lag time and faster growth; therefore, for passages before the weekend, diluting the cell inoculum or using D2%, as mentioned above, are two possible strategies.

46

4.2. Cytotoxicity assays

Several strategies may be used in the screening of extracts or compounds for in vitro cytotoxicity against cell lines. The NCI (National Cancer Institute, U.S.A.) first per-forms a pre-screening against 60 different cell lines representing main tumour types, at a single concentration, 10 μM for pure compounds and 100 μg/mL for extracts (NCI-60 DTP Human Tumor Cell Line Screen). Extracts that produce at least 50% mortality in 3 cell lines are selected for further testing in the presence of several concentrations of the compound.

Another aspect is whether cells are tested in lag or in log phase (Fig. 4.1):

1) Lag phase is the time following subculture and reseeding, in which the cell re-places elements of the glycocalyx lost during trypsinization, attaches to the substrate and spreads out, the cytoskeleton reappears and the cell regains its normal shape. When testing in this phase, the compound is added to the cells when they are seeded.

2) Cells in log phase are exponentially increasing in number, following the lag pe-riod. Adherent cell lines will grow exponentially until they have depleted the me-dium of nutrients or covered the whole surface, at which point growth slows down and reaches a plateau. To perform tests in log phase, cells are first seeded, allowed to attach and begin growing (usually 24 h), and then the compound is added to the cell medium. The NCI Screening Program uses this approach.

Fig. 4.1. Growth phases of cells in culture (adapted from Freshney, 2006).

47

Using 60 different cell lines for routine screening is beyond the budget of most re-searchers, so most bioactivity laboratories will perform these assays with as many cell lines as possible, to detect both cytotoxicity and selectivity.

Carrying out assays in 96 well microplates allows testing different concentrations of the extract or compound. Logarithmic dilutions would be ideal, since dose-response curves are usually characterized by a nonlinear relationship between the compound concentration and the response. However, for practical reasons, two-fold serial dilu-tions are usually carried out. For pure compounds, the highest concentration tested recommended is 40 μg/mL, and for extracts 200 μg/mL.

Cell density and FBS concentration are also important parameters, since they confer protection against cytotoxicity. In the protocol below, 20.000 cells/ well are seeded and FBS concentration is 2%. Cell viability is assessed by the MTT-formazan meth-od, and spectrophotometrically quantifi ed after solubilising the formazan crystals with dimethyl sulfoxide (DMSO).

pROTOCOL 10: Cytotoxicity assays with adherent cell lines

Protocol 9 describes equipment and materials in a more detailed manner. In the other protocols of this chapter, this is dealt with in less detail. Preparation of me-dia and several details about cell line maintenance, such as freezing and thawing, are described in Appendix 3.

Equipment

Incubation chamber with CO 2 and humidity control (37ºC, 5% CO2 and 98% humidity)


Thermostated water bath

Centrifuge

Inverted microscope

ELISA microplate reader with 550 nm fi lter

Vacuum pump with safety fl ask

48



96-well microplates (with modifi ed surface adequate for adherent cell cul-

ture)

Pippeting aids

Sterile pipettes (reusable or disposable)

Sterile Pasteur pipettes (reusable or disposable)

Micropipettes and sterile tips

Multichannel pipette

Repeat pipettor with sterile tips

Neubauer haemocytometer for cell counting

15 mL sterile Falcon tubes in a tube rack

Sterile eppendorfs and eppendorf rack

Container with diluted hypochlorite, to discard media (inside the clean

chamber)

Container with diluted hypochlorite for used materials (outside the clean

chamber)

Flame-sterilizing gas-burner

D2% (DMEM with 2% FBS, antibiotic mixture and other additives) – see

Appendix

D10%

DMSO (dimethylsulfoxide)

EDTA

Trypsin / EDTA

Extract of compound(s) in solvent (DMSO or other), 50 mg/mL (stock

solution/s)

49


Trypan blue

MTT [3 - (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide]

Method

10A. Cells in lag phase

1. Dilute the 50 mg/mL stock solutions in D2% to twice the maximum con-centration to be tested (i.e., 80 μg/mL for pure compounds and 400 μg/mL for extracts).

2. In column 1, pipette 100 μL D2%, using the repeat pipettor. Skip column 2, leaving it empty for now, and pipette 50 μL D2% in columns 3-12.

3. In column 2, pipette 100 μL of the compound solutions prepared in (1). In Fig. 4.2., the microplate is being prepared to test two compounds with four replicates

Fig.4.2 Possible scheme to test two products, with 10 concentrations and four repli-cates each.

4. The serial dilution is carried out with a multichannel micropipette (Fig. 4.3): pipette 50 μL from wells in column 2 and transfer to column 3. Using the mi-cropipette, homogenize by mixing with the D2% already present in column 3 wells (aspire and dispense several times to mix thoroughly).

50


Fig.4.3. Two-fold serial dilution across microplate, from rows 2 to 11.

5. Transfer 50 μL to column 4 and mix thoroughly. Repeat the process until the last column of the dilution series (in the present case, column 11).

6. After homogenizing in column 11, aspire 50 μL and discard to disposal con-tainer.

7. Trypsinize and remove cells from culture fl ask, centrifuge and re-suspend as when passing cells, but using D2% .

8. Homogenize the cell suspension, pipette 50 μL into an eppendorf and add 100 μL Trypan blue. Homogenize gently, to avoid lysis, and count viable cells in an haemocytometer.

9. Dilute cell suspension in D2% to 4x105 cells / mL and homogenize gently with a pipette.

10. Finally, dispense 50 μL of 4x105 cells / mL in D2% with the repeat pipettor to all the rows in columns 2-12 (resulting in 20,000 cells / well and compounds in concentrations 40-0.078 μg/mL, Fig. 4.4).

51


Fig.4.4. Microplate with two compounds in two-fold serial dilutions from rows 2 to 11 (40 to 0.078 μg/ mL), 20,000 cells / well.

11. Incubate in chamber at 37°C, 5% CO2 and 98% humidity for 48h (or longer, if so desired).

12. After 48h, dispense 10μL of MTT into each well, using the repeat pipettor. MTT is light-sensitive, so it is safer to use in unlit clean chamber and protect from excess light.

13. Place in the incubator at 37°C for 3-4h. Remove the microplate from the in-cubator, aspirate the medium with a vacuum pump.

14. With the repeat pipettor, add 100 μL DMSO to each well to dissolve the MTT crystals. Homogenize using the multichannel pipette, or mix in the microplate reader (if the microplate reader model includes this option).

15. Read absorbance at 550 nm (it is possible to use wavelengths from 490 to 600 nm, approximately, although the maximum absorbance MTT-formazan in DMSO is 560 nm).

16. Using column 12 as reference for cell viability, calculate % cell mortality for each concentration and determine EC50 as the concentration which caused 50% mortality.

52


Notes:

The number of concentrations, compounds and replicates tested in a mi-

croplate, depends on the purpose and on the user (e.g., instead of two compounds with ten concentrations, four compounds with only fi ve con-centrations each may be carried out for a rough prescreening).

Rows A and H may also be used as medium blanks and thus receive D2% only.

10B. Cells in log phase

1. Prepare cell suspension as previously, but after centrifuging re-suspend in D10%, Count cells and dilute to obtain 1x105 cells /mL.

2. With the repeat pipettor, pipette 100 μL cell suspension in all columns except column 1 (blank). Place in incubator for 24h (after 24h in D10%, the 10,000 cells /well will have attached and doubled, and therefore there will be approximately 20,000 cells/ well when cells are exposed to the compound).

3. Dilute the 50 mg/mL stock solutions in D2% to obtain the maximum con-centration to be tested (i.e., 80 μg/mL for pure compounds and 400 μg/mL for extracts).

4. In eppendorf tubes, carry out two-fold serial dilutions in order to obtain all the concentrations to be tested.

5. Remove the microplate from the incubator and observe on the microscope to check that cells are in good condition. Back in the clean chamber, aspire the me-dium from all the wells with a Pasteur pipette connected to a vacuum pump. This must be done carefully, avoiding to touch the cells (e.g., by tilting the microplate slightly and aspiring next to the wall of each well).

6. In columns 2-11, pipette 100 μL of the compounds in the appropriate dilutions. Columns 1 (blank) and 12 (untreated cell control) receive D2% only.

7. Place in the incubator for 48h. After this time, proceed as above to assess cell viability and calculate EC50.

53

4.3. determination of mechanisms of cell death by fluorescence

Finding compounds with high cytotoxicity against tumour cell lines is only the first step in the search for molecules with potential chemotherapeutic application. De-termining the mechanism of cell death is extremely important, since it correlates closely with the efficacy, selectivity and magnitude of undesirable side effects. Most molecules used in chemotherapy promote apoptosis, since compounds which cause necrosis will lead to leakage of cell components into the environment, causing in-flammatory processes and damaging healthy tissue surrounding the tumour target. This does not happen in apoptosis, a tightly controlled process in which cytoplasm is retained in apoptotic bodies.

The process of programmed cell death, or apoptosis, is characterized by distinct morphological characteristics and biochemical mechanisms. The detection of apop-tosis therefore can be carried out by identifying the presence of one or more of these characteristics (unambiguous detection of apoptosis requires using more than one technique, e.g., morphological changes, DNA fragmentation, cytochrome c release, caspase activity). During the initial process of apoptosis, cells shrink and pyknosis occurs (condensation of nuclear chromatin). Due to cell shrinkage, cytoplasm be-comes denser and the organelles more compressed. Subsequently, the nuclear enve-lope forms blebs, DNA fragmentation occurs, then the cell is subdivided into several vesicles called apoptotic bodies, which retain cellular materials, which may or not include nuclear fragments, and are finally phagocytosed.

Morphological changes can be detected by conventional microscopy, if very obvi-ous, using conventional staining (e.g., eosin / myosin). These changes are much more obvious when fluorescence stains are used, especially in early apoptosis. This manual will describe the use of nuclear stains DAPI and Hoechst 33342, cytoplas-mic stain BCECF, Annexin-V / FITC, which is used to detect early stages of apopto-sis, and Rodamine 123, which reveals mitochondrial changes which are also indica-tors of apoptosis.

DAPI (4,6-Diamidino-2-phenylindole) is a stain that binds strongly to A-T regions of double DNA, emitting an intense blue color with absorbance maximum at 461 nm and which can be detected with a blue / cyan filter (the emission spectrum is very broad). DAPI crosses cell and nuclear membranes easily and can be used in fixed and in live cells, although the concentration needed for live cells is higher. Due to the process of apoptosis, permeability to the dye is increased and the apoptotic

54

cells will produce a higher intensity of blue fluorescence, which is also increased due to chromatin condensation. Changes in nuclear shape will also become appar-ent, contributing to differentiate between normal and apoptotic nuclei (Fig. 4.5).

Fig. 4.5. Vero cells stained with DAPI. On the left image, normal nuclei can be seen; on the right, nuclear fragmentation, characteristic of apoptosis, is detected. Photos: Vera Gouveia, CIRN lab.

Like DAPI, the Vital dye Hoechst 33342 also binds to DNA and emits at a wave-length of 461 nm. It crosses intact cell membranes and is often used to stain live cells in conjunction with other stains. Nuclear condensation and DNA fragmenta-tion can be detected using this stain, in a similar way as seen for DAPI (Fig. 4.6.).

Fig. 4.6. HeLa cells stained by Hoechst 33342. Pyknotic nuclei, with chromatin con-densation, appear as bright spots. Photos: Vera Gouveia, CIRN lab.

55

The vital dye BCECF is a fluorescent indicator of intracellular pH. BCECF shows excellent retention in cells, and can detect small changes in pH above 7 with a high sensitivity since its pKa (6.97) is ideal near the cytosolic pH of most cells, which renders it ideal as a cytoplasmic stain. In viable cells, BCECF emits an intense green fluorescence (Fig. 4.7).

Fig. 4.7. Vero cells stained with BCECF (green) and Hoechst (blue). On the left, cytoplasm appears intact, whereas on the right only traces can be seen, indicating intense changes in cytoplasmic structure. Photos: Vera Gouveia, CIRN lab.

Phosphatidyl serine exposure on the cell surface occurs in the early stages of apop-tosis and can be identified by staining with annexin V (which is bound to FITC, emitting green fluorescence). Adding Propidium Iodide (PI) which binds to nucleic acids, it is possible to distinguish between early apoptotic, late apoptotic and dead cells. PI cannot cross the cell membrane of viable and apoptotic cells but stains dead cells with red fluorescence. Therefore, in the presence of these two dyes apoptotic cells show a green fluorescence, late apoptotic and dead cells fluoresce red and green, and viable cells show little or no fluorescence (Fig. 4.8).

56

Fig. 4.8. A, HeLa cells stained with Annexin-V /FITC , PI and Hoechst 33342: cells with only blue stain are viable, green circle corresponds to a cell undergoing early apoptosis and red dots are non-viable cells. B, HeLa cells stained with Annexin-V /FITC and PI: green circles are cells undergoing early apoptosis, green and red, cell undergoing necrosis or in late apoptosis, red, non-viable cells. Photos: Vera Gouveia & Duarte Toubarro (A), Duarte Toubarro (B), CIRN lab.

Rhodamine 123 is a membrane-permeable lipophilic cationic fluorochrome which accumulates in mitochondria. It can be used to measure mitochondrial energy en-ergy status, since its accumulation and the resulting fluorescence is proportional to mitochondrial membrane potential. It is also useful for the localization of mitochon-dria in the cell.

Fig. 4.9. Apoptotic HeLa cells stained with Rhodamine 123 (red) and DAPI (blue). A, in non treated cells, the red fluorescence indicating mitochondria is fuzzily distrib-uted across the cytoplasm. B, in apoptotic cells, mitochondria cluster around nuclei, forming semi-circles. Photo: Duarte Toubarro, CIRN lab.

A B

A B

57

One of the features of early apoptosis is the clustering of mitochondria around the nucleus of the cell. Using rhodamine 123 in conjuction with a nuclear-staining dye will reveal this morphological change in cells undergoing apoptosis (Fig. 4.9). Us-ing rhodamine 123 with Annexin V is another possibility, since in this case phos-phatidylserine exposure on the outer cell membrane and mitochondrial clustering will be shown.

pROTOCOL 11: seeding cells for fl uorescence microscopy

Cells are seeded onto polylysine-covered microscope coverslips, placed in wells of a 24-well microplate and allowed to attach and grow for 24 h before being ex-posed to test compound. After staining the cells attached to the polylysine surface, the coverslips can be removed and placed in microscope slides for observation.

Equipment

Fluorescence microscope.


24 well microplate

Polylysine-coated coverslips (these can be prepared as in Appendix 4.6,

or bought pre-coated, as in BD BioCoat™ Poly-L-Lysine 12 mm No. 1 German Glass Coverslips, http://www.bdbiosciences.com/ptProduct.jsp?prodId=364743)

200 µL Micropipette and tips

Eppendorf tubes and rack

D2%

Compound(s) to test in the required concentration

58


Method

1. Place one polylysine-covered coverslip in each well. If possible, leave 1-2 rows free, since they may be used to incubate the cells with the dyes.

2. Add 200 µL of cells in D2% (4x105 cells / mL) and place in the CO2 incubator at 37°C for 24h.

3. After 24 hours, remove the medium (e.g., with a micropipette or a Pasteur pipette) and pipette into the wells 200 µL of medium with the products (e.g., 400 µg / mL for a short exposure or the concentration that is planned).

NOTE: For each treatment time, let a control well with cells and DMEM at 2%, also not forgetting to leave an adjacent empty well.

4. Incubate at 37°C for 12 hours (or less time; have to be tested the appropriate times and concentrations have to be tested for each case; for Annexin V, to detect early apoptosis, 2-6 hours is enough).

pROTOCOL 12: Fluorescence staining protocols

The following protocols were adapted from the instructions supplied by the fl uo-rescent dye manufacturers, therefore they may be changed according to the cell culture type and other conditions in particular.


Micropipettes (200 µL and 2µL) and tips

Poly-prep slides (Sigma P0425)

Fine-tipped tweezers

Distilled water

1X PBS

4% paraformaldehyde in PBS 1X

59


0.1% Triton X-100 in PBS 1X

Gel Mount Aqueous Mounting Medium (Sigma G0918)

500 µg/mL Hoechst 33342 vital dye - prepared by diluting 100 x in PBS,

from a stock solution (50 mg / mL in H2O)

BCECF vital dye - diluted 200 x in PBS from a stock solution (1 mg / mL

in DMSO)

Annexin V Apoptosis Assay Kit # Vybrant 2 (Invitrogen V13241):

1X Annexin Binding Buffer (200 µL Component C + 800 µL o

H2O)

100 μg / mL Propidium Iodide, PI (prepared by diluting dilute 5 µL o

of 1mg/mL PI stock solution, component B, in 45 µL of 1X An-nexin Binding Buffer)

Annexin V conjugate (Component A) o

20 or 10 µg/mL DAPI vital dye (Sigma D9564) – prepared by diluting a

stock solution (1mg/mL in H2O) 50-100x in PBS

100μg / mL Rhodamine 123

Note 1: The dyes have to be kept on ice and protected from light. The proce-dure should be performed in the clean chamber, with the light off.

Note 2: various dyes can be carried out on the same preparation, except DApI, which needs fi xing. In this case, all dyes are added in sequence to the well con-taining the cells (annexin V only works with Annexin Binding Buffer; in this case, this buffer must be used instead of PBS), and incubated for 15-20 minutes in one single step.

60


Methods

12A. hoechst 33342 dye

1. Pipette 200 µL of PBS onto wells that are adjacent to the ones containing cells to stain.

2. With fi ne-tipped tweezers, transfer the coverslips with attached cells into the wells containing PBS.

NOTE: This process must be done very carefully, without turning the coverslip upside down, because the cells must be facing up.

3. Add 2 µL of vital dye Hoechst 33342 (fi nal concentration in the well, 5 µg /mL).

4. Place in incubator at 37°C for 15-30 minutes.

5. Place two small drops of Gel Mount Aqueous Mounting Medium (Sigma G0918) on each side of a microscope slide (not too much, otherwise the coverslip can move and drag the cells).

6. Remove two coverslips from the corresponding wells and place them on the drops, with the cells facing down. Suggestion: it is a good idea to have control and treated cells on the same microscope slide, for comparison.

7. Observe under a fl uorescence microscope with DAPI fi lter (461 nm).

12B. BCECF dye

1. Pipette 200 µL of PBS onto wells that are adjacent to the ones containing cells to stain.

2. Add 1 µL BCECF vital dye (fi nal concentration in the well, 0.1 µM).

3. Place in incubator at 37°C for 15 minutes.

4. Remove the coverslip from the well with PBS and place over Mount Gel Me-dium, as above.

5. Observe in a fl uorescence microscope with the FITC fi lter (488 nm).

61


12C. Annexin V / pI apoptosis assay

1. Remove the coverslip from the well and put it in another containing 200 µL Annexin Binding buffer.

2. Add 4 µL Annexin V conjugate and 2 µL 100μg / mL PI.

3. Place in an incubator at 37°C for 15 minutes.

4. Carefully remove the coverslip from the well and put it in another well with 1X PBS to wash.

5. Remove the coverslip from the well with PBS and place it on the microscope slide with gel mount (just a small drop), as mentioned above.

6. Observe in a fl uorescence microscope with the respective fi lters, Annexin V FITC fi lter (488 nm), PI with the fi lter G 2A (510-560 nm).

12D. DApI dye

1. Remove the coverslip from its well and place it in a solution of 4% paraform-aldehyde (v/v) in PBS to fi x cells.

2. Incubate at 37°C for 20 minutes.

3. Transfer the coverslip to a well with PBS, for washing.

4. Place the coverslip in a solution of 0.1% Triton X-100 in 1X PBS, to permea-bilize the cells.

5. Incubate at 37°C for 15 minutes.

6. Wash coverslip in a well containing PBS; add 2 µL of DAPI vital dye.

7. Leave for 10 minutes, then mount the coverslip in a microscope slide as above, using Gel Mount, and observe on the microscope with DAPI fi lter (461 nm).

62


12E. Annexin V / Rhodamine 123 apoptosis assay

1. Remove the coverslip from the well and put it in another containing 200 µL Annexin Binding buffer.

2. Add 4 µL Annexin V conjugate and 2 µL 100μg / mL Rhodamine 123.

3. Place in an incubator at 37°C for 15 minutes.

4. Carefully remove the coverslip from the well and put it in another well with 1X PBS to wash.

5. Remove the coverslip from the well with PBS and place it on the microscope slide with gel mount (just a small drop), as mentioned above.

6. Observe in a fl uorescence microscope with the respective fi lters, Annexin V with FITC fi lter (488 nm), Rodamine 123 with the fi lter G 2A (510-560 nm).

63

REFERENCES

Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit with Alexa, Fluor 488 annexin V and PI for Flow Cytometry, 2010. Catalog nos. V13241 e V13245, Molecular Probes, Invitrogen

Allen S., Sotos J., Sylte M. J., Czuprynski C. J., 2001. Use of Hoechst 33342 Staining To Detect Apoptotic Changes in Bovine Mononuclear Phagocytes Infected with Mycobacterium avium subsp. paratuberculosis. Clinical and Diagnostic Laboratory Immunology, 8, 460–464

Cell apoptosis DAPI detection kit, Technical Manual No.0358, (www.genscript.com)

Davis, J.M. (Ed) 2002. Basic Cell Culture. Second Edition. A Practical Approach. Oxford University Press, Practical Approach Series, Oxford, 381 pp.

Dukhanin A. S., Patrashev D. V., Ogurtsov, S. I. 1999. Intracellular pH in Thymocytes at the Early Stages of Apoptosis and Necrosis. Bulletin of Experimental Biology and Medicine, 128, 991-993, DOI: 10.1007/BF02433186

Elmore S., 2007. Apoptosis: A Review of Programmed Cell Death. Toxicologic Pathology 35, 495–516

Freshney, R.I. 2006. Basic Principles of Cell Culture. in Culture of Cells for Tissue Engineering, Gordana Vunjak-Novakovic and R. Ian Freshney, Eds., John Wiley & Sons, Inc, pp. 3-22

Hengartner, M. O. 2000. The biochemistry of apoptosis – insight review articles. Nature 407, 770-776

Hotchkiss, R.S., Strasser, A., Jonathan E. McDunn, J.E., Swanson, P.E. 2009.Mechanisms of Disease. Cell Death. The New Engandl Journal of Medicine 361, 1570-83

Houghton, P., Fang, R., Techatanawat, I., Steventon, G., Hylands, P.J., Lee, C.C. 2007. The sulforhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 42, 377–387.

Johnson, L.V., Walsh, M.L., Chen, L.B. 1980. Localization of mitochondria in living cells with rhodamine 123. Proceedings of the Natural Academy of Sciences of the USA 77, 990-994

64

Lagadic-Gossmann D., Huc L. and Lecureur V., 2004. Alterations of intracellular pH homeostasis in apoptosis: origins and roles. Cell Death and Differentiation, 11, 953–961

Lieberman, M.M., Patterson, G.M.L., Moore, R.E. 2001. In vitro bioassays for anticancer drug screening: effects of cell concentration and other assay parameters on growth inhibitory activity. Cancer Letters 173, 21–29

Martinez, M.M., Reif, R.D., Pappas, D. 2010. Detection of apoptosis: A review of conventional and novel techniques. Analytical Methods 2, 996-1004

Masters J. R. and Stacey G. N., 2007. Changing medium and passaging cell lines. Nature Protocols 2, 2276-2284

Nagata S., 2000. Apoptotic DNA fragmentation. Experimental Cell Research 256, 12–8

NCI-60 DTP Human Tumor Cell Line Screen, [http://dtp.nci.nih.gov/branches/btb/ivclsp.html, accessed 5/07/2011]

Niles, A.N., Moravec. R.A., Riss, T.L. 2008. Update on in vitro cytotoxicity assays for drug development. Expert Opinion on Drug Discovery 3, 655-669

Shoemaker, R.H. 2006. The NCI60 human tumour cell line anticancer drug screen. Nature Reviews Cancer 6, 813-823

Subculture of Adherent Cell Lines. Fundamental Techniques in Cell Culture Laboratory Handbook - 2nd Edition (http://www.sigmaaldrich.com/sigma-aldrich/technical-documents/protocols/biology/subculture-of-adherent.html, accessed 7/07/2011)

65

5. Cell-free assays

Maria do Carmo Barreto, Miguel Arruda,Elisabete Rego, Joana S. Medeiros, Nuno Rainha

The search for biological activities in novel compounds is often carried out in cell-free systems, which act as indicators of the existence of a given biological activity in living systems (i.e., cells, tissues and whole organisms). These cell-free systems allow the investigator to (i) monitor the effect of a compound on the rate of a given chemical reaction, or more often (ii) the interaction between a compound and a purified biological target, usually an enzyme or receptor which is involved in the pathway leading to the biological response under study. An example of (i) is the determination of antioxidant activities, where the reduction of an oxidant molecule is usually monitored, or in other cases the protection of a target molecule against oxidative damage is assessed. In (ii) many examples can be cited, such as the inhibi-tion of cyclooxygenases I and II in the search for anti–inflammatory molecules and the inhibition of acetylcholinesterase when testing for molecules which may be used either as bio insecticides or as drugs against Alzheimer’s disease.

Cell-free assays have the advantage of being simpler and cheaper than assays in-volving whole cells or organisms, and are usually less prone to ethical objections. They are also easier to adapt to automated systems and therefore to high-throughput screening. However, they are merely indicative of a putative biological effect, and compounds which yield promising results will always have to be tested in more complex living systems, where metabolism and permeability barriers, among other factors, will determine whether the effect is likely to occur in the desired manner when scaling-up to a whole organism.

66

The present chapter includes protocols for the in vitro quantification of anticholin-esterasic activity and characterization of the type of inhibition, and also protocols for the determination of antioxidant power.

5.1. Interaction with enzymes

Many bioactive compounds act by interfering with an enzyme, changing its activity and causing a metabolic effect. This interference may be an increase in activity or more often an inhibition of the enzyme. Enzyme assays in the absence and in the presence of varying concentrations of the molecules to be tested are carried out. These assays should be carried out in repeatable conditions, and if possible using simple, fast and accurate techniques. Most assays use spectrophotometry or fluor-ometry to monitor the reaction, and if it is possible to adapt the method to use 96-well microplates, the screening becomes faster and requires less amount of reagents and compounds to test.

5.1.1. Inhibition of Acetylcholinesterase

Acetylcholinesterase (AChE, EC3.1.1.7) is an enzyme present in cholinergic syn-aptic junctions which catalyses the hydrolysis of acetylcholine in choline and ace-tic acid, thus terminating neurotransmission. Irreversible inhibitors of this enzyme are extremely dangerous (e.g., sarin gas and several organophosphate insecticides), leading to death by paralysis and asphyxiation. Reversible AChE inhibitors of natu-ral origin, especially when competitive, can be of interest as bio-insecticides, posing far less toxicity problems than traditional insecticides.

Another potential application of reversible inhibitors of this enzyme is in the treat-ment of Alzheimer’s Disease (AD), a neurodegenerative disorder characterized by the reduction of synapses and neuronal death. Most drugs currently used for this disease (e.g., rivastigmine, galanthamine) act by countering the cholinergic deficit associated with cognitive dysfunction and are based on the inhibition of AChE, thus increasing the level of acetylcholine. However, although these drugs contribute to slow down the progress of the disease they cause severe side effects, therefore the search for sources of anticholinesterasic compounds which are both effective, selec-tive and with less negative effects is extremely important.

67

To measure the activity of AChE acetylthiocholine iodide (ATChI), a thiol ana-logue of the natural substrate, is used. The thiocholine resulting from the enzy-matic catalysis of ATChI reacts with Ellman’s reagent (DTNB, 5,5’dithiobenzoic acid ), and the resulting TNB (5-thio-2-nitrobenzoic acid), which is strongly yel-low, is quantified by measuring the absorbance at 415 nm:

ATChI → Acetate + Thiocholine

Thiocholine + DTNB → TNB (yellow)

The following protocols are carried out using purified AChE, therefore the assay is straightforward. When measuring AChE activity in crude extracts one must take into account that Ellman’s reagent reacts with all thiols present in the sample. In this case, a control containing crude extract and DTNB must also be included in the experiment, since GSH, cysteine and other thiols will also contribute to the increase in absorbance. The resulting variation in absorbance can therefore be subtracted from the change due to enzymatic activity.

Compounds that act as strong AChE inhibitors should be further studied, in order to determine the inhibition type and to subsequently characterize the mechanism of interaction with the enzyme.

The first step is to characterize inhibition as reversible or irreversible, since for pharmacological uses reversible inhibitors are preferred. This is easily carried out repeating protocol 12A, but using different times of incubation of the enzyme in the presence of the compound (point 6). If EC50 decreases with incubation time, inhibition is irreversible, if it remains stable, it is reversible.

If inhibition is reversible, it can be characterized as competitive, non-competitive or uncompetitive. This is carried out by determining reaction rates for varying substrate concentrations, without inhibitor and in the presence of at least one concentration of inhibitor molecule.

68

Protocol 13. Inhibition of Acetylcholinesterase

Table 5.1. variation of KM and Vmax with the most common types of reversible inhibition .

Inhibition type parameter change

CompetitiveKMI > KM

Vmax I = Vmax

Non-competitiveKMI= KM

Vmax I < Vmax

UncompetitiveKMI < KM

Vmax I < Vmax

KM and Vmax refer to kinetic parameters in the absence of inhibitor I and KMI

and Vmax I to these parameters in the presence of a given concentration of in-hibitor I.

Diagnosis of inhibition type is based on the variation of kinetic parameters KM (Michaelis constant) and Vmax (maximum velocity) (Table 12.1). In some cases, inhibition cannot be unambiguously classifi ed as one of the categories referred above, and is classifi ed as mixed.

13. A. Determination of IC50

Equipment

Microplate reader with 415 nm fi lter, if possible with temperature control


0.25 U/mL AChE from Electrophorus electricus , e.g., Sigma C2888 , fresh-ly prepared in 100 mM phosphate buffer, pH 7.0, 0.1% BSA ( from a 1000 U/mL stock solution of AChE in 100 mM phosphate buffer, pH 7.0, 0.1% BSA; it is recommended to prepare this stock solution and store aliquots at -80ºC, to avoid freezing and thawing and consequent loss of activity)

69


100 mM sodium phosphate buffer, pH 8.0

3 mM DTNB in 100 mM sodium phosphate buffer, pH 8.0

75 mM ATChI in 100 mM sodium phosphate buffer, pH 8.0

Substrate mixture (prepared by mixing equal parts of 3 mM DTNB and 75

mM ATChI (acetylthiocholine iodide)

5 mg /mL solution of compound or extract in 100 mM sodium phosphate

buffer, pH 8.0 prepared from stock solution (stock solution is prepared in ethanol, methanol or other water-miscible solvent; if possible avoid DMSO or keep it under 0.1% in the fi nal assay, since it inhibits AChE activity); this concentration is only an indication and will have to be opti-mized for each compound (e.g., it is possible for essential oils, which are extremely soluble in ethanol and can therefore allow the preparation of very concentrated stock solutions; pure compounds will usually be tested at a lower concentration range).

96-well microplates

Multichannel micropipette and tips

Repeat pipettor with tips

Multichannel micropipette reagent reservoirs

Method

1. Pipette 240 μL of 5 mg/mL solution of compound into wells in column 2.

2. To the remaining wells (column 1 and 3-12), add 120 μL phosphate buffer.

3. Set the multichannel micropipette to 120 μL, transfer from wells in column 2 to wells in column 3 and mix thoroughly by pipetting in and out. Repeat the procedure until column 11, mix and discard 120 μL from this column after the mixing procedure (Fig. 5.1).

70


Fig. 5.1. Serial dilution from column 2-11. Rows in column 1 are left for blanks and rows in column 12 for control reaction without inhibitor.

4. Add 110 μL phosphate buffer to all the wells in the microplate, using the mul-tichannel micropipette.

5. With the repeat pipettor, add 10 μL 0.25 U/mL AChE to all wells from column 2 to 12.

6. Incubate the enzyme for 5 minutes with the compound.

7. As fast as possible, with the repeat pipettor, add 10 μL substrate mixture to all wells (count time zero from fi rst addition) , place in the microplate reader, pro-grammed to shake for 5 s and read Abs415nm.

8. Read Abs415nm again at times 150 s, 300 s and 450 s.

9. Reaction rates (v, in Δ Abs415nm /min) are calculated using the variation in ab-sorbance from each well with time.

10. Calculate % inhibition of each sample well, using the uninhibited rates from column 12 as control:

%inhibition = 100 – ( v sample / v control ) * 100

71


11. Plot inhibition rates against compound concentration and calculate IC50 as the concentration which causes 50% inhibition of AChE activity (there are several programs that fi t dose-response curves and calculate IC50 and EC50 values, some of them free).

13.B. Characterization of inhibition type

Equipment

Microplate reader with 415 nm fi lter, if possible with temperature control


0.25 U/mL AChE from Electrophorus electricus , e.g., Sigma C2888 , freshly prepared in 100 mM phosphate buffer, pH 7.0, 0.1% BSA ( from a 1000 U/mL stock solution of AChE in 100 mM phosphate buffer, pH 7.0, 0.1% BSA; it is recommended to prepare this stock solution and store aliquots at -80ºC, to avoid freezing and thawing and consequent loss of activity)

100 mM sodium phosphate buffer, pH 8.0

3 mM DTNB in 100 mM sodium phosphate buffer, pH 8.0

75 mM ATChI in 100 mM sodium phosphate buffer, pH 8.0

1.25 mg /mL solution of compound in 100 mM sodium phosphate buffer,

pH 7.0 prepared from stock solution (stock solution is prepared in the ap-propriate solvent, see above); compound concentration must be adapted to each case

96-well microplates




72


Method

1. Prepare S1 substrate solution by diluting 75 mM ATChI with phosphate buf-fer (240 μL 75 mM ATChI + 2760 μL phosphate buffer pH 8.0, therefore yielding a fi nal concentration of 6 mM).

2. Pipette 240 μL of S1 substrate solution into wells in column 2.

3. To the remaining wells (column 1 and 3-12), add 120 μL phosphate buffer.

4. Set the multichannel micropipette to 120 μL, transfer from wells in column 2 to wells in column 3 and mix thoroughly by pipetting in and out. Repeat the proce-dure until column 12, mix and discard 120 μL from this column after the mixing procedure (similar to Fig. 12.1, but until the last column).

5. Add 110 μL of 1.25 mg /mL compound solution to all the wells from column 2 to column 12.

6. Add 10 μL of 3 mM DTNB to all the wells from column 2 to column 12.

7. As fast as possible, with the repeat pipettor, add 10 μL 0.25 U/mL AChE mix-ture to all wells from column 2 to 12 (count time zero from fi rst addition) , place in the microplate reader, programmed to shake for 5 s and read Abs415nm.

8. Read Abs415nm again at times 150 s, 300 s and 450 s.

9. Reaction rates (v, in Δ Abs415nm /min) are calculated using the variation in ab-sorbance from each well with time.

10. Calculate Vmax and KM using appropriate software of by using a standard math-ematical transformation for this effect (e.g., Lineweaver-Burke double reciprocal plot, Eisenthal and Cornish-Bowden’s direct linear plot, or other).

5.2. Antioxidant assays

Oxidative stress is involved in the genesis of many chronic and degenerative disor-ders, such as infl ammation, cancer, cardiovascular, neurodegenerative and age-relat-ed diseases. Oxidation is also responsible for the deterioration of stored foodstuffs, giving rise to the formation of undesirable off-fl avours and unhealthy compounds such as free radicals and reactive aldehydes.

73

In order to prevent oxidative stress and undesirable oxidative reactions in foods, natural or synthetic antioxidants are used. An antioxidant may be defi ned as a sub-stance that, when present at low concentrations compared to those of an oxidizable substrate, such as a lipid, a protein or DNA, signifi cantly delays or inhibits the oxi-dation of that substrate. However, the use of synthetic compounds has been related to health risks resulting in strict regulations over their use in foods. Therefore, there has been an increase in the number of publications related to the investigation of natural antioxidants of natural origin.

Antioxidant assays can be classifi ed according to two general types, namely (1) those associated with lipid peroxidations and (2) those associated with electron or radical scavenging. The fi rst type includes, among others, the thiobarbituric acid and the β-carotene bleaching assays, and the second, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the ferric reducing assays. It is always recommended to perform more than one test when searching for antioxidant compounds, since some molecules give different results according to the assay. These chemical antioxidant assays should be complemented by more complex but also more life-like assays involving live cells subjected to oxidative stress.

pROTOCOL 14: Dpph radical scavenging assay

One of the most widely used methods is based on the reduction of the 1,1 - diphe-nyl-2-picrylhydrazyl radical (DPPH •). DPPH is a stable nitrogen radical, charac-terized by a purple color and an absorbance maximum in the range 515-520 nm, which, when reduced, changes from purple to pale yellow. This method is simple and economic, and only requires a visible spectrophotometer to monitor the de-crease in absorbance due to the presence of an antioxidant. Although DPPH is an artifi cial radical, it became a reference method to assess the antioxidant capacity in vitro thanks to its technical simplicity.

Equipment

Microplate reader with 515 nm fi lter

74



96-well microplates

Micropipettes and tips




Methanol (MeOH)

0.08 mg/mL DPPH in methanol

Freshly prepared standard stock solutions (e.g., 1.0 or 0.50 mg/mL Trolox, Quercetin, ascorbic acid, BHT in the appropriate solvent)

Freshly prepared extract or compound stock solutions in a water-misci-ble solvent (5 mg/mL or less, depending on antioxidant power)

Method

A. Standard

1. For each 96-well microplate, prepare two eppendorf tubes with: (a) a dilution of the standard stock solution (S1: 50 µL standard in DMSO or other + 950 µL of MeOH) and (b) of the solvent (Control: 50 µL of DMSO + 950 µL of MeOH).2. In the microplate, pipette 200 μL of S1 standard solution (50 or 25 μg/mL) , into wells B2, C2 and D2 (Fig. 5.2).

3. Pipette 200 μL of Control solution into wells E2, F2, G2.

4. With the repeat pipettor or with the multichannel micropipette, dispense 200 µL of methanol in all wells from column 1 and 100 μL on all wells from column 3-12.

5. Set the multichannel micropipette to 100 μL, transfer from wells in column 2 to wells in column 3 and mix thoroughly by pipetting in and out. Repeat the procedure until column 12, mix and discard 100 μL from this column after the mixing procedure (Fig. 5.3).

75


6. With the multichannel micropipette or with the repeat pipettor dispense 100 µL of DPPH solution on all wells from columns 2-12.

7. Shake (e.g., use shaking option from microplate reader) and place in the dark for 30 minutes.

8. Read Abs at 515 nm.

Fig. 5.2. Preparing the microplate for the DPPH assay (part 1).

Fig. 5.3. Preparing the microplate for the DPPH assay (part 1, serial dilution). Each column, from 2 to 12, has a concentration equal to half the previous one. The fi rst column is the blank.

76


9. For each well, calculate % antioxidant activity (% AA) as:

% AA = 100 * [(AControl – Asample) / AControl]

Where AControl is the absorbance of the control and Asample is the absorbance of the extract or standard. On rows B, C, D are sample repeats of each concentration, whereas on rows E, F, G, are control repeats for each solvent concentration.

10. Plot antioxidant activity against standard concentration and determine EC50 as the concentration yielding 50% scavenging of DPPH.

B. Extracts

1. Proceed as for the standards, but increase the initial concentration.

2. Results can be expressed as TEAC (Trolox Equivalent Antioxidant Capacity):

TEAC = IC50 Trolox / IC50 sample

pROTOCOL 15: Ferric chloride reduction assay

In this method antioxidant compounds form a colored complex with potassium ferricyanide, trichloroacetic acid and ferric chloride, which is measured at 700 nm. Increase in absorbance of the reaction mixture indicates the reducing power of the samples.

Equipment:

Spectrophotometer

Eppendorf centrifuge

50ºC thermostatic bath or thermoblock

77


Materials and reagents:

0.3 M phosphate buffer, pH 6.6

1% potassium ferrycianide (w/v)

0.1% ferric chloride (w/v)

10% Trichloroacetic acid TCA (w/v)

Extracts in MeOH or DMSO (20-100 μg/mL)

Standards in MeOH, DMSO or H 2O (Quercetin, Trolox, BHT), 100 μg/mL

2 mL eppendorf tubes

Method:

1. In eppendorf tubes, mix 0.4 mL of each extract (or compound) with 0.4 mL of 0.3 M phosphate buffer, pH 6.6 and 0.4 mL of potassium 1% ferricyanide (w/v). Include two “blank” eppendorfs, where extract is replaced by the same volume of solvent (MeOH or DMSO).

2. Incubate the eppendorfs at 50ºC for 20 min..

3. Add 10% TCA to each eppendorf, mix and centrifuge at 11.000 g for 5 minutes.

4. Remove 1 mL of the upper layer of each eppendorf into a spectrophotometer cell.

5. To each spectrophotometer cell add 1 mL H2O, 0.2 mL 0.1% FeCl3.

6. Homogenize and read the absorbance at 700 nm against the blank.

7. Plot Abs700 nm against concentration of extract (or compound).

8. Calculate EC50 by interpolation, using a linear regression analysis, as the con-centration yielding an absorbance value of 0.5.

78

pROTOCOL 16: folin-Ciocalteau total phenolics determination

The Folin Ciocalteau method is one of many used to determine total phenols. This method is based on the reduction of the reagent, a mixture of tungsten and molybdenum oxides. The products of this reduction have a blue color with a broad absorption spectrum, with a maximum at 765 nm. The intensity of light absorption in this wavelength is proportional to the concentration of phenols.

The standard most commonly used in this test is gallic acid (Fig. 5.4) and the concentration of polyphenols is expressed as mg/g GAE (gallic acid equivalent). Since the test quantifi es all polyphenols, the choice of gallic acid as standard comes from the fact that this is a pure and stable substance. It is recommended to prepare a calibration curve every time a determination is carried out.

Fig. 5.4. Chemical structure of gallic acid


Freshly prepared 1:10 dilution of Folin-Ciocalteau reagent

10% Sodium carbonate (w/v)

Standard gallic acid stock solution (5 mg/mL, w/v, in 10% ethanol; it can

be stored at 4ºC for 2 weeks)

Extracts or compounds in the most suitable solvent (usually methanol or

ethanol)

Standard catechin (or gallic acid); use a stock solution with concentra-

79


tion 5 mg / mL, prepared in 10% ethanol and make several dilutions up to 0,050 mg/mL). Note: a stock solution of gallic acid can be stored in the refrigerator for about 2 weeks.

Extracts in the most suitable solvent (note: it is best not to exceed 1-2

mg/mL).

Method

1. Prepare dilutions of the gallic acid standard (e.g., see Table 5.2), for the calibra-tion curve.

[gallic acid] (mg/mL)

Volume (mL)Gallic acid stock solution

(5 mg/mL) H2O

0 0 00.050 0.1 500.100 0.2 1000.150 0.3 1500.250 0.5 2500.500 1.0 500

2. In spectrophotometer cells, add 0.4 mL of 1:10 Folin-Ciocalteau to 0.02 mL of sample (or standard) and 0.2 mL of H2O.

3. After 3 minutes, add 0.8 mL of sodium carbonate solution.

4. Homogenize and incubate for 1 hour at room temperature.

5. Read the absorbance at 750 nm against a blank.

6. For the gallic acid standard curve, plot Abs750 against concentration. Calcu-late the total phenolic content as a gallic acid equivalent (GAE) by interpolation, using a linear regression.

Note: If extracts or compounds absorb at 750 nm, prepare a suitable blank to subtract from the fi nal result.

80

REFERENCES

Alamed, J., Chaiyasit, W., McClements, D.J., Decker, E.A. 2009. Relationships between Free Radical Scavenging and Antioxidant Activity in Foods. Journal of Agricultural and Food Chemistry 57, 2969–2976.

Blois, M. S. 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200.

Brand-Williams, W., Cuvelier, M. E., and Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology 28, 25–30.

Cornish-Bowden, A. 1995. Fundamentals of Enzyme kinetics. Revised Edition. Portland Press, London, 343 pp.

Ellman, G.L, Courtney, K.D., Andres, V., & Featherstone, R.M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88-95.

Giacobini, E. 2000. Cholinesterase Inhibitors Stabilize Alzheimer Disease. Neurochemical Research 25, 1185–1190.

Halliwell, B., 1997. Antioxidants: the basics – what they are and how to evaluate them. Advances in Pharmacology 38:3–20.

Ingkaninan, K.P., Temkitthawon, P., Chuenchom, K., Yuyaem, T., & Thongnoi, W. 2003. Screening for acetylcholinesterase inhibitory activity in plants used in Thai traditional rejuvenating and neurotonic remedies. Journal of Ethnopharmacology 89, 261-264.

Jacobsen, C., Let, M.B., Nielsen, N.S., Meyerb, A.S. 2008. Antioxidant strategies for preventing oxidative flavour deterioration of foods enriched with n-3 polyunsaturated lipids: a comparative evaluation. Trends in Food Science & Technology 19, 76-93

Mattson, M.P. 2004. Pathways towards and away from Alzheimer’s disease. Nature 430, 631-639.

Molyneux, P. 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin Journal of Science and Technology 26, 211-219

81

Monica H., Moisuc A., Radu F., Drăgan S. e Gergen I., 2008. Total polyphenols content determination in complex matrix of medicinal plants from Romania by NIR spectroscopy. Bulletin UASVM, Agriculture 65, 123-128

Moon, J-K, Shibamoto, T. 2009. Antioxidant Assays for Plant and Food Components. Journal of Agricultural and Food Chemistry 57, 1655–1666.

Oki, T., Masuda, M., Furuta, S., Nishiba, Y., Terahara, N., Suda, I. 2002. Involvement of anthocyanins and other phenolic compounds in radical-scavenging activity of purple-fleshed sweet-potato cultivars. Journal of Food Science 67, 1752-1756

Rainha, N., Lima, E., Baptista, J., Rodrigues, C. 2011. Antioxidant properties, total phenolic, total carotenoid and chlorophyll content of anatomical parts of Hypericum foliosum. Journal of Medicinal Plants Research 5, 1930-1940

Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., and Telser, J. 2007. Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry and Cell Biology 39, 44–84.

Waterhouse A.L., 2002. Determination of Total Phenolics. Current Protocols in Food Analytical Chemistry I1.1.1-I1.1.8

83

6. DETERMINATION OF INSECTICIDE ACTIVITY

José Silvino Rosa, Luísa Oliveira, Tânia Teixeira

The increase in agricultural pests has led to a control that is not the most appropriate, due to the incessant use of synthetic insecticides which promote the manifestation of environmental problems. To avoid these problems, research is needed in order to find alternative methods of controlling these pests.

The search for biologically active compounds from natural resources is of great interest to scientists who aspire to the discovery of new features and models for the development of pest control agents that are innocuous to the environment. Particularly interesting in this context are essential oils, which contain active components with insecticidal properties but which present less environmental problems than synthetic pesticides.

There are several methods to test insecticidal activity, such as determining ovicidal activity, contact toxicity or anti-feeding activity. Ovicidal activity is assessed by exposing the eggs of certain insects to the essential oil, extract or compound and quantified by determining the percentage of eggs that did not hatch in relation to the total. The contact toxicity test is based on placing the larvae on a disk of filter paper which was impregnated with the oil. In turn, the anti-feeding activity is estimated by the “deterrence index” caused by treating the diet with the oil. The lower the weight of the diet consumed during treatment (due to the effect of essential oil), the higher the deterrence index and therefore the higher the anti-feeding activity.

84

pROTOCOL 17: Ovicidal activity

Materials

Essential oils from Juniperus brevifolia and Laurus azorica in the fol-lowing concentrations: 1, 10, 100 mg oil / mL of ethanol

Ephestia kuehniella eggs with less than 24 hours

Yellow card (8x70 mm)

Gum arabic solution (30% w/v in water)

Test tubes

Method

1. Put 250 eggs of E. kuehniella on the yellow strips of cardboard (corresponding to an area 40 mm2 (8x5 mm) with the help of gum arabic solution and let dry.

2. Dip the card containing the eggs for 2 seconds, in one of the following solu-tions: water, ethanol, 1 mg oil / mL, 10 mg oil / mL and 100 mg oil / mL.

3. Let dry for 15 minutes before transferring each strip of cardboard into a test tube.

4. Keep the eggs at the appropriate temperature (23ºC).

5. Observe the eggs daily for 6 days and count the number of eggs hatched and not hatched.

6. Calculate the ovicidal activity (OA) by the formula: OA (%) = (number of eggs not hatched / number of total eggs) x 100.

Note: Five replicates should be used for the ovicidal assay.

85

pROTOCOL 18: Contact toxicity

The method of impregnated fi lter paper will be used to assess the contact toxicity.

Material

Essential oils ( e.g., from Juniperus brevifolia and Laurus azorica )

Larvae of Pseudaletia unipuncta in the 4th larval stage

Micropipettes

Petri dishes (5 cm in diameter)

Filter paper

Artifi cial diet for Lepidoptera

Method

1. Place a disk of fi lter paper in a Petri dish.

2. Pipette 100 µL of an essential oil solution (3 mg oil / mL ethanol) on the paper disk and let it dry for 15 minutes.

3. In the control, place only 100 µL of water or ethanol and let it dry for 15 min-utes.

4. Transfer the larvae individually to each Petri dish. The larvae are left without food for 4-5 hours. After this period the larvae are fed, normally, until the end of the test.

5. Keep the larvae at the appropriate temperature (23ºC).

6. Observe and record the mortality during 72 hours. The larvae will be consid-ered dead when they don’t respond to the stimulus caused by an entomological pin. For each treatment, four replicates of 10 larvae are used.

86

pROTOCOL 19: anti-feedant activity

The anti-feeding activity of essential oils will be tested against P. unipuncta in the fourth larval stage by the method adapted from El-Aswad (2003).

Material

Essential oils ( e.g., from Juniperus brevifolia and Laurus azorica ) in the following concentrations: 1, 100 mg oil / mL of ethanol

Larvae of P. unipuncta in the 4th larval stage

Micropipettes

Six-well Elisa microplates

3% Agar

Cut leaf discs of corn ( Zea mays L.) with 7mm diameter.

Method

1. Pre-weigh the larvae, and allow them to starve for 6 hr before the bioassays.

2. Put a little layer of agar in the each well, to prevent desiccation.

3. Dip each leaf disk of corn (with an average weight of 0.0137 mg), for 2 sec-onds, in one of the following: water, ethanol, 1 mg oil/mL or 100 mg oil / ml.

4. After approximately 15 minutes, to evaporate the ethanol, transfer the treated diet to one of the wells of the microplates.

5. Keep the larvae at the appropriate temperature (23ºC).

6. Observe and record the mortality and the diet for 5 days. During this period the larvae are allowed to feed on treated or control diet, respectively.

7. Calculate the anti-feeding activity through the deterrence index,FDI (%) = [(C-T) / (C)] x 100, where C is the weight of the diet consumed in the control and T is the weight of diet consumed in treatment. Each treatment involves three replicates with 10 larvae per replicate.

87

REFERENCES

El-Aswad AF, Abdelgaleil SAM, Nakatani M, 2003. Feeding deterrent and growth inhibi-tory properties of limonoids from Khaya senegalensis against the cotton leafworm, Spodoptera littoralis. Pest Management Science 60, 199–203.

Rosa J.S., Mascarenhas C., Oliveira L., Teixeira T., Barreto M. C. & Medeiros J. 2010. Bio-logical activity of essential oils from seven Azorean plants against Pseudaletia uni-puncta (Lepidoptera: Noctuidae). Journal of Applied Entomology 134, 346–354.

89

AppENDIxES AppENDIx 1


A.1.1. The McFarland Scale

The McFarland scale consists of a series of turbidity standards which represent spe-cific concentrations of Gram-negative bacteria such as Escherichia coli and can be used as a visual estimate of CFU/mL (Colony Forming Units / mL). The standards are prepared by mixing 1% BaCl2 and 1% H2SO4 in the appropriate proportions and are stable for 6 months if stored in the dark at 20-25ºC. Modified standards, using latex bead solutions, have increased storage stability.

Table A.1. Equivalence between McFarland turbidity standards and approxi-mate E. coli concentrations.

McFarland Scale CFU (x 106 /mL) 1% BaCl2 /1% H2SO4

0.5 <300 0.05/5.951 300 0.1/9.92 600 0.2/9.83 900 0.3/9.74 1200 0.4/9.65 1500 0.5/9.56 1800 0.6/9.47 2100 0.7/9.38 2400 0.8/9.29 2700 0.9/9.1

10 3000 1.0/9.0

90

The advantage of this scale is that it requires no equipment or incubation time. The highest disadvantage, besides the obvious subjectivity involved in interpreting the turbidity, is that the numbers are valid for microorganisms similar to E. coli in size and mass. Therefore, before using this method for other microorganisms, a valida-tion and calibration are required.

A.1.2. Correlation between CFU and Absorbance

Although the McFarland scale is widely used, the determination of viable bacterial concentration in CFU / mL (Colony Forming Units / mL) is often required. Correlat-ing absorbance of bacteria in culture medium with CFU provides a simple tool to be used when carrying out the assays. For this effect, growth curves for each bacterial species or strain are carried out, serial dilutions are carried out and plated in solid medium and counted.

Equipment

Laminar flow hood (clean bench) with Bunsen burner•

Spectrophotometer•

Shaker with temperature control (• e.g, termostated bath with shaker)

A.1.2.1. Determination of growth curve

Materials

Inoculating loop•

70% ethanol •

Culture in solid medium•

Liquid broth (• e.g., Tryptic soy broth or other, adequate to the bacterial strain) for pre-inoculum – smaller volume, e.g, in a test tube

Liquid broth for growth curve determination – larger volume, in an erlen-•meyer

91

Petri dishes with solid agar medium•

Method

1. Clean the workspace with 70% ethanol before placing materials in laminar flow hood.

2. Flame-sterilize inoculating hoop, allow it to cool (e.g., by placing it in the agar of the culture Petri dish) and remove bacterial culture into the pre-inoculum liquid broth.

3. Place at growth temperature (e.g., 30-37ºC) with shaking, overnight.

4. Remove an adequate volume from pre-inoculum into erlenmey-er with liquid broth (inoculation volume will vary with bacterial species and strains, e.g, 1 mL pre-inoculum/ 100 mL liquid broth in final culture). 5. Homogenize and remove 0.5 mL to measure Abs600nm at time zero, and place cul-ture in the shaker, at the adequate growth temperature.

6. Remove 0.5 mL of bacterial culture at intervals (e.g., every hour) and measure Abs600nm, diluting with broth when necessary to obtain absorbance < 1, until station-ary phase is attained (i.e., when absorbance values practically stop increasing.

7. Plot growth curve of the bacterial culture (Abs600nm as a function of time, see Fig. A.1).

Fig. A.1. Example of a bacterial growth curve. Cell growth is monitored by the abo-sorbance at 600nm.

92

A.1.2.2. Determination of CFU

Materials

Inoculating loop•

70% ethanol •

Culture in solid medium•

Liquid broth (• e.g., Tryptic soy broth or other, adequate to the bacterial strain) for pre-inoculum – smaller volume, e.g, in a test tube

Liquid broth for growth curve determination – larger volume, in an erlen-•meyer

Petri dishes with solid agar medium (a 100 mm Petri dish needs approxi-•mately 20-25 mL of agar medium)

6 Test tubes with 9 mL nutrient broth•

1. From the growth curve plotted with the results, choose one or two points in the middle of the exponential growth phase (in Fig. A1, around 8-12 h after inocula-tion).

2. Prepare a bacterial culture as above, and let it grow until it reaches exponential phase. At the chosen time(s), remove 1 mL to monitor Abs600nm and 1 mL to a sterile tube containing 9 mL of nutrient broth.

3. Homogenize and remove 1 mL to the following tube. Repeat until 10-6 dilution (Fig. A.2).

Fig. A.2. Serial dilution and plating to determine correlation between Abs600nm and CFU.

93

4. From each dilution, plate 0.1 mL onto three Petri dishes containing nutrient agar, spreading well.

5. Place at the appropriate temperature for 8-12 h and count the colonies.

6. Plot CFU as a function of Abs600nm.

Note 1: Each isolated colony is theoretically originated from a single bacterial cell, so a number of viable cells (or CFUs) can be calculated for each dilution

Note 2: It is advisable to count colonies in plates containing between 20 – 200 colo-nies; a small number may have no statistical significance, whilst a higher number of colonies will originate errors and colony overlap.

AppENDIx 2

Jorge Humberto Leitão

A.2.1. Nematode growth media

NGM I (Nematode Growth Medium I (Stierngale, 1999) – for normal growth

NaCl 1.5 gTryptone 1.25 gAgar 8.5 gH2O (distilled) adjust to 500 mLNystatine (10 mg/ mL) 2.5 mLAdditives (see below)

94

NGM II (Nematode Growth Medium II, Tan et al., 1999) –for assays

NaCl 1.5 gBactopeptone 1.75 gAgar 8.5 gH2O (distilled) adjust to 500 mLNystatine (10 mg/ mL) 2.5 mLAdditives (see below)

Additives

1 M KPO4 buffer, pH 6.0 12.5 mL1 M CaCl2 0.5 mL1 M MgSO4 0.5 mLUracil (2 mg /mL, sterilized by filtration) 0.5 mLCholesterine (10 mg/mL in ethanol) 0.25 mL

Nystatine solution (10 mg/ mL)

Nystatine (-20ºC) 4 gEthanol (96%) 200 mLAmmonium acetate solution 200 mLMix, sterilize by filtration, and store at -20ºC

Ammonium acetate solution

Ammonium acetate 173.4 gH2O (distilled) adjust to 300 mL

95

1 M KpO4 Buffer, ph 6.0

KH2PO4 108.3 gK2HPO4 35.6 gH2O (distilled) adjust to 1000 mL

and pH 6.0

M9 Buffer, ph 6.0

KH2PO4 3 gNa2HPO4 6 gNaCl 5 g1 M MgSO4 1 mLH2O (distilled) adjust to 1000 mL

and pH 6.0

Autoclave all additives separately and autoclave media and buffers. Wait until me-dia are cooled down to approximately 50ºC before adding additives.

Pour NGM I into Petri dishes with 5.5 cm diameter (Greiner) and NGM II to into 6-well plates (4 mL / well, Greiner)

A.2.2. Maintenance of C. elegans – freezing and thawing

1. Prepare an egg suspension according to Protocol 7.

2. Pour the egg suspension on the surface of NGM I plates without E. coli OP50.

3. After 1-2 days, rinse L1-larvae with 1 mL PBS buffer and centrifuge 1 minute (2000 rpm or 800 g).

4. Discard the supernatant and wash the pellet additionally three times in PBS buf-fer.

5. Finally, resuspend the pellet in 800 µL PBS buffer, mix with 200 µL of 30%

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glycerine and transfer to a cryo tube. Wait for 1 hour at room temperature before freezing at -80 ºC.

6. When the eggs are needed, thaw the culture for 30 minutes at room temperature, mix shortly with 0.5 mL PBS buffer and wait for further 30 minutes at room tem-perature. Repeat the procedure.

7. Use several drops of the culture, mixed with PBS buffer and pipette it on a NGM I plate (with E. coli OP50).

8. Observe the plate for several hours, add PBS buffer if necessary.

AppENDIx 3

Laila Moujir Moujir, Maria do Carmo Barreto

A.3.1 Cell line suppliers

Cell lines, derived from cancers or from normal tissue, human or otherwise, can be obtained from several suppliers. The ATCC (American Type Culture Collection) has the highest number of cell lines, and can be reached at http://www.atcc.org. The ECACC (European Collection of Cell Cultures), although smaller, is also a valu-able resource: http://www.ecacc.org.uk. Among other suppliers, cell lines can also be obtained at the German DSMZ (http://www.dsmz.de) and the Japanese JCRB (http://cellbank.nibio.go.jp/) . Most suppliers will send the cell cultures either fro-zen or in culture, according to request.

A.3.2. Media and materials for mammalian cell culture

Several firms supply media, reagents and materials for cell culture. Most of the media and reagents below are from Sigma, although in many cases other suppliers supply comparable ones, with the same quality standards (e.g., GIBCO / Invitrogen). Ob-

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viously, all manipulations must be carried out in sterile environment, i.e., in a clean chamber. Choosing between sterilizing some materials by autoclaving or using only disposable sterile materials will depend on a balance between the budget and the risk of contamination. The same can be said for several media and reagents which can be either prepared and sterilized in the lab, or bought sterile and ready to use.

1. Supplementing DMEM culture medium:

DMEM (Dulbecco’s Modified Eagle’s Medium), Sigma D6546, contains most nu-trients and factors needed by mammalian cells. However, when opening a new bot-tle, several supplements may be added:

freshly prepared (or freshly thawed) L-glutamine is routinely added to the

medium, since its half-life at 4ºC does not exceed 3 weeks

antibiotics and antimycotics may be added to the culture medium, although

ideally it would be better to avoid them, since they may eventually alter the biochemistry of the cells

1A. preparation of complete DMEM:

For 1 L DMEM add:

10 mL Antibiotic mixture (S/P)

1 mL Antimycotic

10 mL L-Glutamine

Stir thoroughly, without inverting

Store at 4ºC

1B. Supplementing complete DMEM with FBS (e.g., D2%, D5%, D10%)

Fetal bovine serum (FBS) is often added, since it provides several factors,

some of which promote cell growth and adhesion.

We recommend preparing FBS-supplemented DMEM in smaller amounts,

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in sterile screw-cap bottles, from Complete DMEM and the amount of in-activated FBS required for the desired concentration (label: D2%, D5%, D10% or other unambiguous labeling system used in the lab)

Store at 4ºC.

2. Trypsin / EDTA

Trypsin /EDTA may be prepared in the lab and sterilized by filtration, or

bought (e.g., Sigma T4049, supplied as a frozen sterile solution).

To avoid contamination and loss of activity, it is good practice, when open-

ing a new bottle, to divided it by aliquots (e.g., 10 mL each, in 15 mL sterile Falcon tubes labelled T/E) and storing at -20ºC.

3. FBS inactivation

Many authors recommend this step, in which fetal FBS is heated to 56 °C in a

water bath to destroy heat-labile complement proteins prior to use in cell growth medium, since these proteins might contribute in some degree to cell lysis:

Thaw FBS at 37ºC in a water bath with a level slightly higher than the level

in the bottle

Raise the temperature of the bath to 56ºC; during this incubation, invert the

bottle to mix the serum every 10 min

When the bath reaches 56ºC, incubate serum for 30 min; invert bottle every

10 min

Remove FBS bottle from bath and allow to cool

Aliquot into 15 mL conical tubes (Falcon), 8-10 mL / tube and store at

-20ºC.

4. antibiotic mixture - streptomycin / Penicillin (s/P)

2.5 g Streptomycin sulphate (Sigma S6501, powder, ≥720 units /mg)

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1.51 g Penicillin G sodium salt (Sigma P3032-10MU, powder, ≥1477 units /mg)

Add distilled water until 25 mL

Sterilize by filtration with a 0.22 μm filter

Aliquot into 15 mL conical tubes (Falcon) labelled S/p, 5 mL each, and store at -20ºC

5. Antimycotic – sodium hydroxybenzoate (hB)

10 mg sodium 4-hydroxybenzoate (Sigma H3766, >98% purity)


Sterilize by filtration with a 0.22 μm filter

Aliquot into 15 mL conical tubes (Falcon) labelled hB, 5 mL each, and store at 4ºC

6. pBS (phosphate Buffer Saline, concentrated) 10 x

80 g NaCl

2g KCl

11.87 Na 2HPO4 (anhydrous)

2 g KH 2PO4 (anhydrous)

Add distilled water until 1 L

Sterilize by autoclaving

Aliquot in 200 mL portions, in sterile screw-cap bottles (Schott)

Store at 4ºC

7. pBS 1 x

200 mL PBS 10X

100

Add distilled water until 1 L



Store at 4ºC

8. 1% phenol Red

2.5 g Phenol Red

81.25 mL 0.1 N NaOH




Store at 4ºC

9. 0.02% EDTA (Ethylenediaminetetraacetic acid)

0.1 g EDTA (free acid)

50 mL PBS 10X

0.75 mL 1% Phenol Red



Aliquot in 40 mL portions, in conical tubes (50 mL Falcon)

Store at -20ºC

10. 200 mm l-glutamine

14.615 g L-Glutamine


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Sterilize by filtration at 0.2 μm, 0.45 μm prefilter

Aliquot in 40 mL portions, in conical tubes (50 mL Falcon)

Store at -20ºC

11. mtt (3-(4.5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide)

0.1 g MTT

20 mL PBS

Sterilize by filtration with 0.2 μm filter, protecting from light (MTT is light

labile)

Aliquot in tubes wrapped in aluminium foil

Store at 4ºC

A.3.3. Freezing and thawing cell cultures

Using cells which are growing rapidly will increase the percentage of viability after freezing / thawing. Therefore, cells must be frozen just as they become confluent. One way to achieve this is to prepare 100 mm dishes or equivalent surface area (e.g., T50 flask), give them fresh medium the day before freezing and freeze them just as they become confluent. DMSO is also added as a cryoprotector, however, since it is highly toxic at room temperature, it has to be added with care (adding it slowly helps to avoid osmotic shock). FBS is often also added to increase cell viability. Another important aspect is to ensure that temperature decreases slowly; this can be achieved using a freezing box containing isopropanol and the cryotube rack (e.g., Nalgene 5100 Cryo 1°C Freezing Container, “Mr. Frosty”, claims to “provide the critical, re-peatable -1°C/minute cooling rate required for successful cell cryopreservation and recovery”); however, placing the cells in a polystyrene box with some insulating material will also work (in our lab, we place the cryotubes in polystyrene box, which in turn is kept inside a bigger polystyrene box filled with cottonwool). Freezing is carried out in two or three steps: cells are first placed at -20ºC, then at -80ºC and finally, for long-term storage, in liquid nitrogen. Cells at -80ºC, if properly frozen, will retain viability for several months.

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Freezing box (either a proper freezing box containing isopropanol, or a

polystyrene box with some insulating material)

Standard material to work with cell culture

D10%, freshly prepared

D20%, freshly prepared

Freezing medium (20% DMSO in D20% )

A. Freezing

1. Tripsinyze an almost confluent 90-100 mm Petri dish or equivalent (one T50 flask, two T25 flasks…).

2. Centrifuge in D10%.

3. In a cryotube, re-suspend the pellet in a very low volume of D20% , in order to obtain a high cell density (for a 90-100 mm Petri dish, approximately 0.5 mL D20%).

4. Very slowly (dropwise), add 0.5 mL of feezing medium to the re-suspension, swirling after each addition. A compromise must be attained between adding freez-ing medium slowly, but not taking too much time, since DMSO at room tempera-ture may damage cells.

5. Place in a pre-cooled freezing box and place at -20ºC for 2.5h.

6. After 2.5 h, change the freezing box to -80ºC and leave overnight.

7. If possible, put the cells in liquid nitrogen (now only the cryotube is needed, since the cells are now fully frozen). As mentioned before, cells stored in liquid nitrogen will last indefinitely retaining viability (at least in theory).

Note: some researchers use FBS-free freezing medium.

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B. Thawing

1. Place thawing medium (D20%) at 37ºC (e.g., in an eppendorf tube floater, in the water bath).

2. Remove the cryotube from the freezer and thaw the cells at 37ºC.

3. Carefully, with a Pasteur pipette, transfer the thawed cells onto a 15 mL conical tube (Falcon).

4. Add approximately 6 mL D20%, dropwise, to avoid any risk of osmotic shock, carefully swirling the tube to homogenize gently.

5. Centrifuge at 200 g for 5 minutes.

6. Re-suspend with 1 mL D10%, homogenizing carefully by pipetting in and out. Add 4 mL more D10% and homogenize likewise.

7. Place in a small culture flask (T25) or 30 mm Petri dish.

8. Five hours later, check that cells have begun to attach by observing at the micro-scope. If they have, eliminate medium with non-attached dead cells and replace with fresh D10%.

Note: some researchers avoid the centrifugation step, and directly dilute the thawed cells with warm D20%, adding it dropwise in a conical tube and then transferring the cell suspension into a Petri dish.

A.3.4. Counting cells in a haemocytometer


Eppendorf tubes and rack

Trypan blue

Neubauer haemocytometer

1. After trypsinizing and re-suspending cells, if cell counting is needed, extra care is

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necessary to homogenize by the suspension pipetting in and out of a pipette.

2. In an eppendorf tube, add 50 μL homogenized cell suspension to 100 μL Trypan blue. Homogenize carefully, to minimize cell lysis.

3. Transfer a drop into the Neubauer chamber with a coveslip. The area under the coverslip fills by capillarity (Fig. A.3)

Fig. A.3. Neubauer haemocytometer (adapted from Alcântara et al., 2001).

4. In the microscope, with low magnification, count the cells that are not stained blue. (Trypan blue will only enter cells with damaged cell membrane, therefore blue cells are non viable). Count 3-5 counting grids (large squares, see Fig. A.4).

Fig. A.4. Central area of Neubauer haemocytometer.

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5. Using the average count of 3-5 squares (“mean value”), calculate cell number as:

Cell number= (mean value)x3x104 cells /mL

6. Dilute as required for the assay or other use of the cell suspension.

AppENDIx 4

Maria do Carmo Barreto, Vera Gouveia, Duarte Toubarro

a.4. Coating growth surfaces with polylysine (for fluorescence or other micro-scopy observation of cells)

Polylysine coverlips can be prepared in the lab or bought pre-coated (e.g., BD BioCoat™ Poly-L-Lysine 12 mm No. 1 German Glass Coverslips, http://www.bd-biosciences.com/ptProduct.jsp?prodId=364743, accessed 12/07/2011).

Materials and solutions

microscope coverslips

1% HCl in 70% ethanol

Distilled H 2O

0.1% Polylysine (w/v), diluted 1:10 in distilled water

rack to dry coverslips

diamond tip scriber ( i.e., a pen with a diamond tip used to write on glass)

fine-tipped tweezers

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Method

1. Wash each coverslip in a beaker containing 1% HCl in 70% ethanol, holding in with the tweezers.

Fig. A.6. Sequence of beakers containing solutions to coat microscope coverslips with polylysine.

2. Wash the coverslip in distilled water (beaker 2).

3. Place the coverslip in beaker 3, containing polylysine, and leave for 5 minutes.

4. Remove the coverslip from beaker 3 and place in a drying rack.

5. Repeat the same procedure with each of the lamella.

6. Place overnight in an incubator at 60 ºC.

7. When coverslips are dry, cut them with a diamond tip so they can fit in the wells of the 24 microplate wells (buying round coverslips with the right size avoids this step).

8. Sterilize coverslips by autoclaving wrapped in aluminium foil (or under UV).

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