FRET Analysis of STIM1-Orai1 Interaction

HEK293 cells were transfected with 3 µg STIM1‐mCherry‐pmax (containing mCherry after position L599 (Alansary et al, 2016 (link)) or STIM1A‐mCherry‐pmax (acceptor) and 1µg Orai1‐GFP‐pmax (donor) 24 h before measurements). Prior to the measurements, TG‐sensitive stores were depleted using 1 µm TG in 0 [Ca²⁺]_o Ringer. For recording fluorescence images, the Leica AM TIRF MC system was used. Images were taken with a 100 × 1.47 oil HCX Plan Apo objective. The GFP‐Donor signal was used to determine the TIRF focal plane. For each cell, three images were taken: I. GFP excited at 488 nm (suppression filter BP 525/50); II. mCherry excitation wavelength at 561 nm (suppression filter BP 600/40); and III. FRET excitation with a 488‐nm laser and suppression filter BP 600/40. To calibrate laser intensities and excitation durations for each day of experiments, single transfected cells were used, only expressing the donor or acceptor construct. Calibrated parameters were constant for all channels and images taken. For image acquisition and analysis, the LAS (Leica Application Suite) FRET module was used. To calculate FRET efficiency according to van Rheenen et al (2004 (link)), all images were corrected for background signal, bleed‐through, and crosstalk factors.