Visualization and Quantification of Actin Dynamics in Rice Protoplasts

Materials and Reagents

1. Rice protoplasts
   
2. Plasmid pUN1301-EGFP-ABD2-EGFP (Yang et al., 2011)
   
3. D-Mannitol (Sigma-Aldrich, catalog number: M1902 )
   
4. 2-(N-Morpholino) ethanesulfonic acid (MES) (Merck KGaA, catalog number: 475893 )
   
5. Cellulase “Onozuka” RS (Yakult Pharmaceutical Industry, catalog number: 9012-54-8 )
   
6. Macerozyme R-10 (Yakult Pharmaceutical Industry, catalog number: 8032-75-1 )
   
7. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
   
8. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C5670 )
   
9. β-mercaptoethanol (Merck KGaA, catalog number: 444203 )
   
10. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
    
11. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
    
12. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M2393 )
    
13. PEG4000 (Merck KGaA, catalog number: 817006 )
    
14. Murashige and Skoog basal salt mixture powder (MS) (Sigma-Aldrich, catalog number: M5524 )
    
15. Digestion buffer (see Recipes)
    
16. W5 buffer (see Recipes)
    
17. Mmg buffer (see Recipes)
    
18. Polyethylene glycol solution (see Recipes)
    

Equipment

1. 35 μm nylon mesh
   
2. Spinning disk confocal microscope equipped with a Yokogawa CSU-X1 spinning disk head using a 512 x 512 Andor iXON electron-multiplying CCD camera (Andor)
   
3. Shaker
   
4. Centrifuge
   
5. Concave slide (diameter 15 mm) (Beyotime, catalog number: FSL011 )
   
6. Olympus BX61 inverted microscope equipped with a 100x 1.4 NA UPLSAPO (Universal Plan Super Apochromat Objective) objective
   
7. 488 nm laser
   
8. 525/50 nm band-pass filter
   

Software

1. Andor IQ2 software (available at http://www.andor.com)

Procedure