The kinetics of the formation of the Orai1 clusters at the PM plane was evaluated using TIRF microscopy on cells transiently transfected with equimolar amounts of Orai1-YFP and STIM1-CFP. Imaging was performed at room temperature on a Zeiss Cell observer TIRF system fitted with a 63×/1.46 lens using the following parameters: λex = 488 nm, filter for λem = 510 to 555. The frame rate was 0.2 Hz and the typical exposure time was 50 ms. TIRF images in the PM plane were thresholded and normalized to the levels before TG to empty the stores. Puncta formation was assessed by increased fluorescence in the TIRF plane.