Nk92 cells

The genetically encoded, mitochondria-targeted pScalps_CEPIA2mt calcium reporter was transduced into NK92 cells (American Type Culture Collection, CRL-2407), sorted by flow cytometry, and used to measure mitochondrial calcium flux via flow cytometry. To measure the cytosolic calcium levels, these NK92 cells were also loaded with 2 µM Indo-1-AM (Thermo Fisher Scientific) in the presence of Pluronic F-127 (Sigma-Aldrich) and 2.5 mM probenecid (Thermo Fisher Scientific) in HBSS (Life Technologies) with 2 mM calcium and 2% FCS for 30 min at 37°C. Subsequently, full α-MEM was added and incubated for another 30 min at ambient temperature to enable intracellular trapping of Indo-1. The loaded cells were surface stained with CD45-BV785 (BioLegend) and LIVE/DEAD near-infrared marker (Thermo Fisher Scientific) and washed once. Samples were kept on ice and warmed in a 37°C water bath for 3 min prior to acquisition. Data were acquired on a FACSymphony A5 (BD Biosciences) equipped with a UV laser. Calcium flux was triggered by adding MK6-83 or bafilomycin A1 as indicated in the graphs. Ionomycin (1 µM) was added at the end of the assay to show maximum responsiveness of the system. CEPIA_mt fluorescence and the ratiometric Indo-1 fluorescence were collected in the FACSDiva flow cytometer (BD Biosciences), and the FCS files were exported to FlowJo (BD Biosciences) for further analysis.

The genetically encoded, mitochondria-targeted pScalps_CEPIA2mt calcium reporter was transduced into NK92 cells (American Type Culture Collection, CRL-2407), sorted by flow cytometry, and used to measure mitochondrial calcium flux via flow cytometry. To measure the cytosolic calcium levels, these NK92 cells were also loaded with 2 µM Indo-1-AM (Thermo Fisher Scientific) in the presence of Pluronic F-127 (Sigma-Aldrich) and 2.5 mM probenecid (Thermo Fisher Scientific) in HBSS (Life Technologies) with 2 mM calcium and 2% FCS for 30 min at 37°C. Subsequently, full α-MEM was added and incubated for another 30 min at ambient temperature to enable intracellular trapping of Indo-1. The loaded cells were surface stained with CD45-BV785 (BioLegend) and LIVE/DEAD near-infrared marker (Thermo Fisher Scientific) and washed once. Samples were kept on ice and warmed in a 37°C water bath for 3 min prior to acquisition. Data were acquired on a FACSymphony A5 (BD Biosciences) equipped with a UV laser. Calcium flux was triggered by adding MK6-83 or bafilomycin A1 as indicated in the graphs. Ionomycin (1 µM) was added at the end of the assay to show maximum responsiveness of the system. CEPIA_mt fluorescence and the ratiometric Indo-1 fluorescence were collected in the FACSDiva flow cytometer (BD Biosciences), and the FCS files were exported to FlowJo (BD Biosciences) for further analysis.