A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors

Table 4 Quantities of stock solutions and reagents required for the preparation of working buffers for wheat and barley mesophyll leaf protoplast isolation and transfection

Reagents/stock solutions	Protoplast isolation buffer^a	Wash buffer^a	Transfection buffer 1^a	Transfection buffer 2^a	Regeneration buffer^a
0.8 M Mannitol	7.5 ml		12.5 ml	1.25 ml	11.25 ml
PEG 4000				2 g
0.1 M MES pH 5.7	1 ml	1 ml	1 ml		600 µl
2 M KCl	100 µl	125 μl			150 µl
5 M NaCl		1.54 ml
1 M MgCl₂			375 µl
Cellulase R10	150 mg
Macerozyme R10	50 mg
Heat to 55 °C for 10 min, cool to room temperature
1 M CaCl₂	100 µl	6.25 ml		500 µl
BSA	10 mg
ddH₂O	Up to 10 ml	Up to 50 ml	Up to 25 ml	Up to 5 ml	Up to 15 ml
	Filter sterilize	Filter sterilize			Filter sterilize