Bimolecular Fluorescence Complementation Assay

For BiFC, single wells in the U96 Microwell plate were used to transfect protoplasts with plasmids encoding proteins of interest fused to the N-terminus of nEYFP and cEYFP. Empty vector (3 μg) encoding Enhanced Cyan Fluorescent Protein (ECFP) was added to each well as a transfection control. After transfection, protoplasts resuspended in 200 μl of W5 solution with 5 mM glucose [33 (link)] were transferred to black 96-well black glass-bottom plates (SensoPlate, Greiner Bio-One, www.greinerbioone.com), sealed with transparent sealing tape (Thermo Scientific Nunc, cat. no. 236366), and returned to the growth chamber for overnight incubation. After incubation, the sealing tape was removed and the plate was screened with a Nikon A1Rsi confocal system (www.nikon.com). See Additional file 8 for original files.