Protoplast Preparation and Determination of Cell Death

Materials and Reagents

1. Plant materials: Arabidopsis thaliana sterile seedlings grown in vitro from 1 to 3 weeks in the required conditions
   
2. Gamborg B5 medium salt and vitamins (Duchefa Biochemie, catalog number: G0210 )
   
3. 2-(N-Morpholino) ethanesulfonic acid (MES)
   
4. Mannitol
   
5. Digestion enzymes
   1. Cellulase (Duchefa Biochemie, catalog number: C8001 )
      
   2. Macerozyme (Duchefa Biochemie, catalog number: M8002 )
6. Culture medium (see Recipes)
   
7. Digestion medium (see Recipes)
   
8. W5A medium (see Recipes)
   
9. 21% sucrose (see Recipes)
   
10. 1% Evans blue (see Recipes)

Equipment

1. Light microscope
   
2. An appropriate slide (e.g. Malassez slide)
   
3. Centrifuge for 15 ml and 50 ml Falcon tubes
   
4. Sterile pipette
   
5. Filter (0.45 µm)
   
6. Hemocytometer (VWR International, catalog number: 631-0975 )
   
7. Cell dissociation sieve (100 µm) (Sigma-Aldrich, catalog number: CD1-1KT ) sieve mounted on a sterile 150 ml beaker
   
8. Micropore tape (VWR International, catalog number: 115-8172 )
   

Procedure