Fluo 4 direct kit

Calcium imaging experiments were performed as described previously (Deering-Rice et al. 2011 (link), 2012 (link); Shapiro et al. 2013 (link)). Briefly, cells were loaded with Fluo-4 AM using the Fluo-4 Direct Kit (Invitrogen) diluted in LHC-9 (HEK-293 cells) or calcium assay buffer (HaCaT cells) for 60 min at 37°C (HEK-293) or room temperature (HaCaT). The loading solution was removed and the cells were subsequently incubated 30 min at 37°C (HEK-293) or room temperature (∼23°C) (HaCaT) in the dark with LHC-9 (HEK-293) or calcium buffer (HaCaT) containing 0.5 mmol/L probenecid and 0.75 mmol/L trypan red (ATT Bioquest, Sunnyvale, CA). Treatment solutions were prepared in LHC-9 (HEK-293) or calcium assay buffer (HaCaT) at 3× concentration and 10 or 25 μL was added to 20 or /50 μL of media on the cells in 384- or 96-well plates. Calcium flux responses were measured using either a NOVOstar plate reader (BMG LABTECH, Offenberg, Germany) or microscopically as described previously in the references above. Where specified, data were corrected for nonspecific responses observed with HEK-293 cells, and normalization to the maximum attainable change in fluorescence elicited by ionomycin (10 μmol/L).