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

Materials and Reagents

1. 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)
   
   
2. 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
   
   
3. 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 )
   
   
4. 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)
   
   
5. 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

1. 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
   
   
2. 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

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

Procedure