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www.europ-continents.com

Flow Cytometry Application: AgroscienceBalai Penelitian Tanaman Pemanis dan Serat (Balittas)

Europ Continents IndonesiaEurop Continents IndonesiaPARTEC –pioneer in flow cytometry–

Part of Sysmex corporation since 2013

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A method for counting, examining, and sorting microscopic particles suspended in a stream of fluid

It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus

Europ Continents IndonesiaEurop Continents IndonesiaFlow Cytometry

5/12/11

Flow Cytometry

Sheath fluid

Sample(stained cells in suspension)

NozzleHydrodynamic FocusingCells pass through in ‘single file’

Fluorescence emitted from stained cells detected

Forward and side scattered light from all cells detected

Laser light source

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FluidicsHydrodynamic focusing

OpticsScatters & fluorescence channels

ElectronicsDigitized data

InformaticsSpecialized software

Europ Continents IndonesiaEurop Continents IndonesiaParts of Flow Cytometer

The heart of flow cytometer

Stained sample

*Particle size range : 0,2 - 200 µm

ApplicationApplicationMain CategoriesMain Categories Sub CategoriesSub Categories RemarksRemarksHealthcare Immunology

Stem Cell Pathology Cell Culture Apoptosis Sorting

Partec offers flexibility for multicoloranalysis with 1 or several lasers.

HIV, Solid tumor analysis, DNA analysis,Cell Cycle analysis, Ploidy measure-ment, DNA/Protein Ratio, DetectionOf minor DNA variations, PRINS, FISH,

etc

MicrobiologyMicrobiology Bacteria Yeast Biotechnology Virus & sub-micron particle

detection Research in microbiology

antimicrobial/antibiotic analysis, bacteria counts, population dynamics and quantification of pathogens, Enumeration of growing cells, quantification of cellular yield, detection of contaminations with undesired cells, , etcetc

AgrosciencesAgrosciences Agrosciences, Animal & wildlife Agrosciences, Animal & wildlife research, Aquaculture, Plant research, Aquaculture, Plant breedingbreeding

Ploidy analysis, Hybridization, Apomixis, Ploidy analysis, Hybridization, Apomixis, Genome size, Effect of Endoreduplication, Genome size, Effect of Endoreduplication, sperm viability, immuno status, etcsperm viability, immuno status, etc

Industrial Application

Food & Beverages, Milk & Dairy, Personal care / cosmetics, Pharmaceuticals, Paper industries.

TVC, Total Yeast & Mould, Pathogen test, particle test, hydrophobic contaminants, , hydrophobic contaminants, etc

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Komoditas Tanaman Balittas

o Tanaman pemanis (tebu, bit, stevia) o Tanaman serat buah (kapas dan kapuk), o Tanaman serat batang (rami, kenaf, rosela, jute, abaca,

agave, linum, dan tanaman serat batang yang lain)o Tembakauo Tanaman minyak industri (wijen, jarak kepyar,

jarak pagar, kemiri minyak, dan bunga matahari)

Application FCM- Agroscience

o Detection of Ploidy Levelo Determination DNA content in absolute unit (genome size)o Detection of Hybridso Cell cycle analysiso Determination of sex in plantso Detection of mutation plantso Pollen viability

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FCM for Measurement of plant DNA

General FCM Sample Preparation1. Put fresh leave sample in clean petri dish

2. Add extraction buffer

3. Chop the sample with sharp razor blade

4. Incubate for 1-5 min

5. Remove large debris, by filter the sample through Partec Celltrics

6. Labeling : add the staining solution

DAPI (4',6-diamidino-2-phenylindole)

PI (propidium iodide)

DAPI used in UV LEDPI used in green or blue laser

Ploidy level detection (in Sugarbeet)

Nuclear DNA content correlates with ploidy

Rapid, Convenience, and non destructive

Function: Control of ploidy stability, ploidy screening, interspecific hybrid, aneuploid detection

Sample: Tissue chopping or lysis of protoplast

Diploid sugarbeet

Small young Leaves

1 mL buffer Tris+ DAPI

Isolation of nuclei by chopping the tissue

Add 2-3 mL pure water & filtered

40µm mesh

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Ploidy level detection (in Jathropa curcas)

Nuclear DNA content correlates with ploidy

CRBC as external std (diploid = 2C)

Diploid

20-30 mgSmallyoung leaves

600 µL buffer LB01 supplemented w/ 100

mM citric acid

Isolation of nuclei by chopping the tissue

Filter through 30µm mesh, add 2µg/ml DAPI

Flow cytometric analysis

• Flow cytometric analysiso f DAPI stained J. curcas nuclei in suspension leaf nuclei

Ploidy analysis of regenerated plantlet (in Ramie)

• Sample : shoot regeneration of plantlet from cotyledon of ramie (Boehmerianivea Gaud)• Diploid ramie as control standard

Diploid

1,5 cm² young Leaves

400µL extraction buffer

Isolation of nuclei by chopping the tissue

Filtered through 30µm mesh + DAPI stain

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Determination DNA content in sugarcane

• Sample : Saccharum officinarum cv.

Green German

• Zea mays (2C DNA content = 5,43 pg) as internal standard

0.5 cm² leaf tissue + Zea mays (int.std)

0,5 ml buffer lysis

Isolation of nuclei by chopping the tissue

Removal of large debris by filtration + DAPI staining

Flow cytometric analysis

Flow cytometric analysis

Isolation of nuclei by chopping the tissue

Determination DNA content in cotton• Sample : cotton (Gossypium hirsutum)

• Oryza sativa (2C DNA content = 1,01 pg) as external standard• Machine analyzed for 5,000–10,000 nuclei were per sample

1,5 cm² leaf tissue + 1,5 mL buffer lysis

0,5 ml buffer lysis

Removal of large debris by filtration (50µm mesh)+ staining sol.(PI +Rnase)

O.sativa

G.hirsutum

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Determination DNA content in Jute• Sample : Corchorus olitorius (Tossa jute)

•The genus Corchorus contains phenoliccompounds and several other secondary metabolites

• Supplemented with 1% PVP 25 and 0,1% Tween20 to improve the histogram results

• Radish (Raphanussativus) as internal std

15 mm² cotyledons + 20 mm² radish (int.std)

0,7 ml buffer lysis

Isolation of nuclei by chopping the tissue

Removal of large debris by filtration + PI staining sol (PI + Rnase).

Incubate 30min

Flow cytometric analysis

Intraspecific Hybrid Screening sugarcane (Saccharum spp.)

When parental species differ enough in their nuclear DNA content, flow cytometric analysis can detect interspecific hybrids according to their intermediate DNA values

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• Peak 1 : S. officinarum cv. Green German• Peak 2 : S. spontaneum cv. IND 81-146• Peak 3 : US99-1003, a n+n progeny of the Green German x IND 81-146 cross with an intermediate DNA content • Corn (2C = 5.43 pg) as internal standard

c

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Intraspecific Hybrid Screening cotton Somatic hybrids were produced through protoplast electrofusion between the

tetraploid cotton Gossypium hirsutum L. cv. ZDM-3 and the wild diploid cotton G. klotzschianum

Flow cytometry analysis of relative DNA content of leaf nuclei : a ) G. hirsutum cv. ZDM-3 (parent)b ) G. klotzschianum (parent)c ) representative of somatic hybrid

Hybrid had chromosome numbers near to sum of the two parents (78 = 52 + 26)

Production of fertile somatic hybrids of Gossypium hirsutum + G. bickii via protoplast fusion

Flow cytometry analysis of relative DNA content of leaf nuclei. (a) G. hirsutum L. cv. Coker 201 (parent); (b) G. bickii (parent); (c) representative of somatic hybrid.

DiploidG.bickii

TetraploidG.hirsutum

Hexaploid :Hybrids

• G.hirsutum as standard adjusted to channel 100 (tetraploid)• G. bickii showed a DNA content of 65 channels (diploid)• Each histogram was generated by the analysis of at least 10,000 cells and repeated four times• DNA content of hybrids : 161-167, fusion between 4x and 2x protoplast

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Cell cycle analysis

DNA content reflects the position of a cell within a cell cycle

(A) : untreated culture

(B) : treated culture with drugs that effect the cell cycle distribution and induces apoptosis (Ap)

Automated cell cycle button in CyFlow space®

Effect of CycD1 expression in the tobacco (Nicotiana tabacum) cell suspension culture BY-2, in cell cycle progression

* Used a modified AlcR/AlcA two-component gene expression system that permits inducible gene expression in cell cultures in response to added dexamethasone (Dex) to monitor the consequences of D1 cyclin gene expression.

Samples (5mL) centrifuged 3 min, 700 rpm

Pellets were immediately frozen in liquid nitrogen and kept at80°C before analysis.

The frozen pellets (;100 to 200 mg) were thawed with gentle agitation in 300mL of Partec extraction buffer. Add 1,5 mLstaining buffer

Filter 30µm mesh

Flow cytometric analysis

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Determination of Sex in Plants

Sex chromosomes are differ in size and hence in DNA content.

X chromosome (female) has lower DNA content than Y-chromosome (male).

The G1 and the G2 peaks corresponding to both sexes are clearly separated

The assay sex determination would give benefit in area where rapid sex identification is required at early stage of plant identification.

Analysis of Effects of Irradiation/ mutation plants

The increasing doses of X-rays, effect in variation of nuclear DNA content

Results : nuclear DNA content was influenced by different doses of X- rays

With increasing the dose, nuclear DNA content was decreased.

The decrease in nuclear DNA content was directly related to the plant genome size.

The ploidy level are not affected by irradiation even at higher dose

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Pollen viability

Using Pollen viability Kit from Partec

Protocol :

- Suspend 50.000- 300.000 pollen grains in PBS in sample tube

- Add 10 μl of Pollen Viability Solution A to the test tube and incubate for 10 min at room temperature

- Add 10 μl of Pollen Viability Solution B to the test tube

- Analyse in blue laser (488nm), emission green (FL-2) and red (FL-3) fluorescence

Instruments Recommended

Picture Model Description

CyFlowSpace

Partec CyFlow Space• Customized to needs /

applications : • 5 parameter (FSC, SSC, 3

colour fluorescence)• Light source : UV-LED,

Blue laser 20mW

CyFlow PA

Partec CyFlow PA :• UV LED, 1P• Green laser, 2P• UV LED & Green laser, 2P

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CUSTOMER CITY TYPE/ UNIT MODEL INSTAL APPLICATION No. Kontak

BB- BIOGEN Bogor PARTEC CCA 2002 Ploidy & Apoptosis

Telp: 0251 -8338820,83334408345975

BLK Jayapura Jayapura CyFlow SL-3 2007 CD4 – CD4% (0967) 534304

BASF Chemical Jakarta CyFlow SL-5 2007 Research

PT East West Seed Indonesia

Purwakarta CyFlow Space UV 2008 Ploidy Level 0264-201871- 4

PT LondonSumatera

Medan CyFlow Space UV+Blue

2009 Ploidy Level, pollen viability,hybrids

Telp. (061) 4532300

BLK Ambon Ambon CyFlow Counter II 2009 CD4 – CD4% 0911-348652

PT LondonSumatera

Medan CyFlow PA 2009 Ploidy Level Telp. (061) 4532300

RS Tarakan Tarakan CyFlow Counter II 2010 CD4 – CD4% 0551-51745

PT LondonSumatera

Medan CyFlow PA (2 unit) 2010 Ploidy Level Telp. (061) 4532300

PARTEC Flow Cytometry References in Indonesia

CUSTOMER CITY TYPE/ UNIT MODEL INSTAL APPLICATION No. Kontak

RS Freeport Timika CyFlow Counter II 2011 CD4 – CD4% 0971-7666

RSUD RantauParapat

Medan CyFlow Counter II 2011 CD4 – CD4% (0624) 21228

LIPI Cibinong Cibinong CyFlow Space 2011 Research 021-87907604/ 87907636

RS Marzuki Mahdi Bogor CyFlow Counter II 2012 CD4 – CD4% 0251-8320467

Dinkes PemkabMadiun

Madiun CyFlow Counter II 2012 CD4 – CD4% (0351) 464242 (0351) 466437

Dinkes PemkabKuningan

Kuningan,Jawa Barat

CyFlow Counter II 2013 CD4 – CD4% (0232) 871149

PT SMART, Tbk Bogor CyFlow Space 2013 Research

PT Kemira Indonesia Cikarang Cyflow Cube 6 2014 Sticky control monitoring

RS Paru Batu, Malang Malang CyFlow Counter II 2014 CD4 – CD4%

PT Tolan Tiga Medan Cyflow PA 2014 Ploidy analysis

Agronomi-Univ.Hassanudin

Makassar Cyflow space-5P 2015 Research

PARTEC Flow Cytometry References in Indonesia

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Contact Us

PT. Europ Continents IndonesiaT: 021-8313743/44 F : 021-8314137

Email: lifescience.jkt@europ-continents.com