Detecting ER Calcium Dynamics in Cells

The ER-GCaMP6-150 plasmid, provided by T.A. Ryan (de Juan-Sanz et al., 2017 (link)), is a variant of GCaMP6 Ca²⁺ indicator containing the signal peptide of calreticulin and KDEL ER retention motif suitable for detecting Ca²⁺ in the ER. WT abl preB cells carrying Tet-On Cas9 transgene were infected with lentivirus carrying ER-GCaMP6-150. Cells emitting green fluorescence were isolated by flow-cytometric cell sorting and single-cloned. The responsiveness of ER-GCaMP6-150 to Ca²⁺ fluctuation in the ER was confirmed by flow-cytometric analysis following treatments with 50 nM SERCA inhibitor thapsigargin for 30 min. The levels of ER-GCaMP6-150 fluorescence in the cycling abl preB cells expressing gRNA were analyzed on day 3 or 4 of doxycycline treatment. For cytosolic Ca²⁺ analysis, abl preB cells (without ER-GCaMP6-150) were treated with 2 µM Fluo 3-AM (Sigma-Aldrich; 39294) at 37°C for 30 min in growth medium. The stained cells were washed once in prewarmed growth medium and then aliquoted for thapsigargin treatment before flow cytometry or analyzed directly.