Screening of GCaMP and NCaMP7 variants in HEK293 cells. b , Ca 2+ imaging-based screening. A typical trace from NCaMP7-expressing cells is shown; a.f.u. arbitrary fluorescence units. To avoid saturation of the camera, after recording the basal fluorescence ( F 0 ) with regular exposure time (approximately 500 ms), time-series for variants with high dynamic range were recorded using one-tenth to one-fifth the exposure time. Afterwards, the fluorescence response curves of each cell were scaled up according to the corresponding F 0 . After recording F 0 , endoplasmic reticulum Ca 2+ store was depleted using 2.5 M iono and 1 M TG. The cells were then incubated in an imaging solution containing 300 M EGTA, to read minimal GECI fluorescence ( F min ). Finally, the cells were exposed to imaging solution containing 100 mM Ca 2+ to obtain the maximal response ( F max ) via SOCE.
