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Mammalian Cell Culture and Western Blotting

Summer Training ReportSubmitted toJawaharlal Nehru UniversityByPallavi Raj Sharma

Summer School - 2015School of Life SciencesJawaharlal Nehru UniversityNew Delhi 110067Acknowledgement

I thank Prof. B. C. Tripathy, Dean, SLS, for granting me the opportunity to attend Summer School-2015 in this esteemed institution.

I am grateful to Dr. Nirala Ramchiary, Dr. Neelima Mondal and Prof. P. C. Rath for being easily approachablefor resolving our queries and organizing this Summer School.

I would like to thank Prof. Rana. P. Singh, School of Life Sciences, Jawaharlal Nehru University, for his guidance and encouragement. I am privileged to have worked under his supervision.

I am highly grateful for motivation, support and hospitality of my seniors in lab. I sincerely thank Dr. Ajay and Ms. Reenu for their extended involvement and encouragement which made my experience in lab worth the while. I would also like to thank Ms. Lalita, Mr. Mathan, Mr. Mohit, Ms. Nidhi, Mr. Praveen, Ms. Saba and Mr. Vijay for their stimulating presence, problem solving and intriguing discussions.

I would like to express my heartfelt gratitude to the entire faculty of School of Life Sciences, Jawaharlal Nehru University for the insightful lectures, which always sparked a curiosity to know more.

Above all, I would like to thank my parents, friends and family for their support. Their constant encouragement and faith in me has always inspired me to keep moving forward and give my best in all my endeavors.

Thank you, God, for blessing me with all that I have.

Pallavi Raj Sharma

Abbreviations

rpmRevolutions per minute

FBSFetal Bovine Serum

P/SPenicillin-Streptomycin

PBSPhosphate Buffer Saline

DMSODimethyl Sulphoxide

ddH2ODouble distilled water

SDSSodium Dodecyl Sulphate

PAGEPolyacrylamide Gel Electrophoresis

ODOptical Density

APSAmmonium Persulfate

TEMEDTetramethylethylenediamine

ppmParts per million

psiPounds per square inch

CONTENTSPage no.1. Cell Culture1a. Experiment 1 : Revival3b. Experiment 2 : Splitting3c. Experiment 3 : Cryopreservation5d. Experiment 4 : Treatment5

2. Western Blotting6a. Experiment 5 : Extraction of whole cell lysates6b. Experiment 6 : Bradford Assay8c. Experiment 7 : SDS-PAGE10d. Experiment 8 : Electrotransfer12e. Experiment 9 : Immunoblotting13

3. Cell Culture Contamination154. Discussion185. References19

1. CELL CULTURECell culture is the method of culturing or maintaining cells procured from human tissues in vitro, i.e. in an artificial environment providing all essential nutrients and conditions optimally. The cells can be procured either directly from a human biopsy sample or tissue by enzymatic or mechanical methods or can be revived from a cryo-preserved vial containing frozen cells. Cryopreservation is the process of storing cells at -196oC in liquid nitrogen, which freezes the cells in their present state and inactivates all metabolic processes. This initial inoculation of cells on an artificial growth media is referred to as primary culture. The cells are provided with the correct temperature, gaseous tension, pH and nutritional requirements, and thus they spread all over the plate. Once the cells achieve 80-90% confluency, they need more space and nutrients to grow further, thus need to be subcultured or passaged. Subculturing is the process of removing cells from the primary culture plate and introducing them into a fresh media plate. Many secondary plates can be prepared from a single primary plate.While subculturing, the following numerical terms need to be considered:1. Passage number: This refers to the number of times a cell line has been transferred to a fresh plate. The cell line quality degrades with each passage, sometimes show genetic drift, hence there is a limit to the number of subcultures you can carry out. In such cases, the student must cryopreserve the cultures at a low passage number and work with one until the maximum passage limit is reached. 2. Split ratio: Each cell line has a specific split ratio that guides us about how many secondary plates can be derived from one primary cell culture plate. For instance, a split ratio of 1:4 indicates the confluent primary culture cells can be used to initiate 4 new secondary culture plates.Media The growth media for optimum growth of inoculated cells consists of a standardized mixture of essential nutrients, growth factors and hormones along with regulating the pH and the osmotic pressure of the culture. Basal Media contains amino acids, vitamins, inorganic salts, and a carbon source such as glucose.Serum is an important component of culture media as it is a source of growth and adhesion factors, hormones, lipids and minerals which enhances the growth considerably. In addition, serum also regulates cell membrane permeability and serves as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell. Although addition of serum is highly beneficial for good cell growth, there are some problems concerned with their use including high cost, problems with standardization, specificity, variability, and unwanted effects such as stimulation or inhibition of growth and/or cellular function on certain cell cultures. Serums can also be a source of contamination, therefore must be checked before adding to the media.The growth media is thus usually supplemented with 10% serum and 1% antibiotics.pHFor optimal growth of cells in artificial medium, an optimum pH of 7.4 must be maintained. pH can be checked visually by adding few drops of media on pH test strips. Phenol red changes color with change in pH.CO2Buffers provide resistance against pH changes in cell cultures and help maintaining a constant pH. The buffering is usually achieved by including an organic (e.g., HEPES) or CO2-bicarbonate based buffer. The pH of the medium is prone to change with change in external CO2 as it depends on the balance between dissolved CO2 and bicarbonate ions in the buffering system. Hence a specific oxygen tension needs to be maintained and 410% CO2 is common for most cell culture experiments. However, each medium has a recommended CO2 tension and bicarbonate concentration to achieve the correct pH and osmolality. This CO2 tension is maintained by a CO2 incubator where the cells are kept for incubation at 37oC.Temperature Most human and mammalian cells are maintained at 37C for optimal growth. Experiment 1Aim: Thawing and revival of cryopreserved cellsMaterials required: Cryovial containing frozen cells, growth medium, pre-warmed to 37C, 15ml falcon tubes, Water bath at 37C, 70% ethanol, tissue-culture platesProcedure:1. The cryovial was taken out from liquid nitrogen storage and kept in a pre-warmed 37C water bath.2. The cells were allowed to thaw completely by swirling it and the vial was taken to the biosafety cabinet.3. 1ml of thawed cells was added to 2ml of growth media in a falcon. 4. The cell suspension was centrifuged at 1000-1200 rpm for 510 minutes. A pellet of the cells was obtained, and the supernatant was discarded without disturbing the pellet. 5. The cells were resuspended in 3ml complete growth medium and transferred to a culture plate and kept in incubator.Observations: The cells were successfully revived.----------------------------------------------------------------------------------------------------Experiment 2Aim: Splitting revived cells SubculturingMaterials required: Complete growth medium (RPMI 1640) (900ml), FBS (100ml) and P/S antibiotic (10ml), sterile petriplates, Trypsin, 1X PBS, pipettes, hemocytometerProcedure:1. The spent media which now appears yellow-orangish due to acidity of cell metabolites was taken out using a pipette and discarded carefully without disturbing the adhered cells.2. The cells were washed twice, with 2ml PBS or media and swirled then discarded.3. 1ml Trypsin was added to the petriplates and incubated in the CO2 incubator at 37oC for 5 minutes.4. The cells were observed under the microscope for detachment. 5. 2ml complete media was added to inactivate Trypsin and the detached cells were dispersed in the fresh media by pipetting in and out a few times.6. The falcon tube was then centrifuged at 1000-1500 rpm for 5 minutes.7. The supernatant was discarded and the pellet obtained of the cells was resuspended in 5ml complete media and mixed uniformly using a pipette.8. 10ul from the cell suspension was put in a clean hemocytometer, the cover slip was put on and observed under microscope.9. The number of cells was counted in each 4X4 box and noted down. The number of cells per ml was calculated by multiplying the average cell count with 104. Likewise, calculation was made for the volume of suspension containing 3 lakh cells.10. The required volume was taken and complete media was added so as to make 10ml final volume.11. 1ml each from this suspension was added to fresh plates containing 8-10ml complete media and mixed uniformly be sliding in a zig-zag fashion.12. The plates were checked under the microscope for uniformity and then kept in CO 2 incubator. Observations: After approximately 24-36 hours, the sub-cultured cells were observed to be adhered and achieved 70-80% confluence.

ba Fig 1. Subcultured A549 cells observed under 40X (a) and 100X (b) magnification.Experiment 3Aim: Cryopreservation of cellsMaterials required: Cultured cells, Typsin, DMSO, FBS, cryovialProcedure:1. The spent media was taken out using a pipette and discarded carefully without disturbing the adhered cells.2. The cells were washed twice, with 2ml PBS or media and swirled then discarded.3. 1ml Trypsin was added to dislodge the cells and a cell suspension was formed.4. The cell suspension was centrifuged and the supernatant discarded.5. The pellet was resuspende