FACS法进行拟南芥精子和营养细胞核的样本制备和分离

Nina Chumak; Magdalena Mosiolek; Vera Karolina Schoft

doi:10.21769/BioProtoc.1664

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Peer-reviewed

Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS

FACS法进行拟南芥精子和营养细胞核的样本制备和分离

Nina Chumak

MM Magdalena Mosiolek

Vera Karolina Schoft email

发布: 2015年11月20日第5卷第22期 DOI: 10.21769/BioProtoc.1664 浏览次数: 12953

评审: Samik BhattacharyaYurong XiePengpeng Li

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Mar 2015

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Abstract

One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 10⁶ sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.

Keywords: SYBR Green (荧光染料掺入法)

Fluorescence-activated cell sorting (FACS) (荧光激活细胞分选（FACS）)

Arabidopsis (拟南芥)

Pollen (花粉)

Male gametophyte (雄配子体)

Materials and Reagents

Plastic ware / consumables
1. 15 ml polypropylene tubes (SARSTEDT AG & Co, catalog number: 62554502 )
2. 50 ml polypropylene tubes (SARSTEDT AG & Co, catalog number: 62548004 )
3. 100 µm nylon mesh (Biologix Group Limited, catalog number: 151100 )
4. 40 µm nylon mesh (Biologix Group Limited, catalog number: 151040 )
5. 250 ml centrifuge tubes (Bartelt, catalog number: 9.315 721 )
6. 1.5 ml and 2 ml microliter reaction tubes
7. Syringe filters 0.2 µm (Bartelt, catalog number: 9055511 )
8. BD Falcon^® 12 x 75 mm tube with cell strainer cap (BD Biosciences, catalog number: 352235 )
  Note: Currently, it is “Corning Inc., catalog number: 352235”.
9. Sterican disposable hypodermic needles with a diameter of 0.45 x 25 mm (B. Braun Melsungen AG, Braun Petzold, catalog number: 4657683 )
10. Pipettes tips (1,000 µl, 200 µl, 20 µl, 2 µl)
11. Acid-washed glass beads (see Recipes)
Glassware
1. Glass beads 0.4-0.6 mm (Sartorius AG, catalog number: BBI8541701 )
2. 250 ml Erlenmeyer flask
3. Glass tubes
4. Glass slides
5. Cover slips
Plant material
1. 100 Flowering wild-type Arabidopsis thaliana plants (Col-0 accession and dml-3 mutant, are used as examples) (Arabidopsis, catalog number: SALK_056440 )
Chemicals
1. D(+)-Sucrose for analysis (AppliChem GmbH, catalog number: 131621.0914 )
2. DAPI (4', 6-Diamidino-2-Phenylindole, dihydrochloride) (Life Technologies, Invitrogen^TM, catalog number: D1306 )
  Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: D1306”.
3. SYBR Green I nucleic acid stain stock solution (Life Technologies, catalog number: S7567 )
  Note: Currently, it is “Thermo Fisher Scientific, Invitrogen^TM, catalog number: S7567”.
4. Phenylmethanesulfonyl fluoride (Sigma-Aldrich, catalog number: 93482 )
5. Complete, EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11873580001 )
6. Calcium chloride dihydrate (AppliChem GmbH, catalog number: A3587 )
7. Ethylenediamine tetraacetic acid disodium salt dihydrate (Carl Roth GmbH + Co., catalog number: 8043 )
8. Ethylene glycol-bis (2-aminoethylether)-N, N, N, N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
9. Ficoll^® PM 400 (Sigma-Aldrich, catalog number: F4375 )
10. Glycerol (Sigma-Aldrich, catalog number: G5516 )
11. Magnesium acetate tetrahydrate (Sigma-Aldrich, catalog number: M5661 )
12. Sodium chloride (EMD Millipore Corporation, catalog number: 106404 )
13. Potassium chloride (Sigma-Aldrich, catalog number: P3911 )
14. D-Sorbitol (Sigma-Aldrich, catalog number: S6021 )
15. Spermine [N, N'-Bis-(3-aminopropyl)-1, 4-diaminobutan] (AppliChem GmbH, catalog number: A0672.0005 )
16. Triton^TM X-100 (Sigma-Aldrich, catalog number: 234729 )
17. Tris-base (AppliChem GmbH, catalog number: A1086 )
18. Nitric acid 65% (Carl Roth GmbH + Co., catalog number: X898.1 )
19. Hydrochloric acid fuming 37% (Carl Roth GmbH + Co., catalog number: 4625.1 )
20. Sodium Hydroxide pellets (AppliChem GmbH, catalog number: A3910 )
21. Ultrapure water (MilliQ) (conductivity > 18 MΩ^-1 cm^-1)
Buffers
1. BD FACSFlow Sheath Fluid (BD Bioscience, catalog number: 342003 )
2. Sucrose solution (see Recipes)
3. Buffer A (see Recipes)
4. Buffer B (see Recipes)
5. Protease inhibitor stock solution (see Recipes)
6. DAPI stock solution (see Recipes)

Equipment

Instruments
1. Beckman Coulter Avanti J-26XP centrifuge with the JA-10 rotor
2. Table-top centrifuge
3. Orbital shaker (VWR International, catalog number: 89032102 )
4. Drying oven (EHRET GmbH, catalog number: 3108 )
5. Bead-beater (Retsch GmbH, model: MM301 )
6. Fluorescence microscope with 20x, 40x, and 100x objectives and a DAPI filter
7. Flow cytometer: FACS Aria I (BD Biosciences) with a 70 µm nozzle and 70 psi sheath pressure, and a 488 nm coherent sapphire solid state 13 mW laser for excitation and a 530/30 nm band-pass filter for detection of SYBR green I
General lab equipment
1. Beaker
2. 250 ml Erlenmeyer flask
3. Glass tubes
4. Sharp-edged scissors
5. Forceps
6. Air displacement pipettes
7. Ice container

Software

Flow cytometric software (BD Biosciences, model: FACS Diva 6.1.2)

Procedure

English

中文翻译

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Chumak, N., Mosiolek, M. and Schoft, V. K. (2015). Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS. Bio-protocol 5(22): e1664. DOI: 10.21769/BioProtoc.1664.