Intact hSS at day 43–52 were incubated with 10 μM Fluo-4 acetoxymethyl ester (Fluo-4AM; Invitrogen) for 30 min in NM media followed by a 15 min wash with NM. A Leica SP8 confocal microscope with a resonant scanner was used for imaging. Spontaneous calcium activity was recorded for 10 min (one frame every 8–10 s) in one 10 μm z-stack plane. Fluorescence intensity (F) was exported as mean gray values in ImageJ. Signal decay was controlled by subtracting the mean fluorescence of the background (Fb). To estimate changes in intracellular calcium, ΔF was computed as (Fcell– Fb)/F0, where F0 represents the minimum F value per cell across the whole 10 min of recording from which Fb was subtracted. A ΔF >1.2 was defined as a spike.
