Fig. 9From: A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptorsOverview of steps for the transfection of isolated protoplasts. Step 18: 300 µl Transfection Buffer 1 containing protoplasts are transferred to each transfection tube. Step 19: Using a pipette, plasmid transfection sample 1 is added to transfection sample tube 1 directly into Transfection Buffer 1 containing protoplasts. Step 20: 350 µl Transfection Buffer 2 is added to transfection sample tube. Step 21: Transfection sample tube is inverted 12 times. Step 22: Transfection sample tube is placed into rack in the dark. Step 23: Steps 19 to 22 are repeated with all other transfection samples one after another. Step 24: After 15 min incubation in the dark, 2× 660 µl of Wash Buffer is added to transfection sample tube. Step 25: Transfection sample tube is inverted 8 times. Step 26: All transfection sample tubes (up to six at the time) are centrifuged at 100×g for 3 min. Step 27: Using a pipette, 2× 965 µl are removed from all transfection sample tubes. Step 28: 965 µl of Regeneration Buffer is transferred into each transfection sample tube. Step 29: All transfection samples tubes containing protoplasts are regenerated at 20 °C in the dark for 16 hBack to article page