Cd45 bv785

Freshly isolated or ex vivo cultured human cells were washed in PBS with 5% bovine serum (FACS buffer) and subsequently stained for 30 minutes at 4°C with fluorophore conjugated antibodies against CD19, CD20, CD22, CD27, or IgD (all BD Biosciences). Cells were then washed twice in FACS buffer and analysed on a FACS Canto2 (BD Biosciences). Where appropriate dead cells were excluded using 4,6-Diamidin-2-phenylindol (DAPI; Thermo Fisher).
To analyse B cells after 9–11 days in culture, plates were spun at 400×g for 3 minutes, supernatants were taken for analysis by ELISA and cells in pellets were re-suspended and pooled for each condition. Cells were washed with PBS and stained with NIR fixable live/dead dye (Molecular probes) for 15 minutes at room temperature and washed with FACS buffer (2%BSA, 2mMEDTA, PBS). Live/dead staining was followed by an incubation with Fc Block reagent (BioLegend) for 10 minutes at RT and finally staining with a mix of labelled antibodies: CD45-BV785, CD19-FITC, CD27-BV711, CD38-APC, CD138-PE, and IgD-BV421 (all BioLegend) following the manufacturers recommendations for 30 minutes at 4C. Stained cells were washed and fixed with 2% PFA in PBS for 10 minutes at RT. Stained cells were analysed in a Beckton Dickinson Fortessa with 355, 405, 488, 561, and 633nm lasers.

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 following antibodies were used in flow cytometry: B220 FITC 1:50 (BD Pharmingen, RA3-6B2), CD3 BV421 1:200 (17A2, BioLegend), CD3 FITC 1:50 (17A2, BD Pharmingen), CD3 PE-Cy-7 1:25 (145-2C11, BD Pharmingen), CD11b PerCP-Cy5.5 1:400 (M1/70, eBioscience), CD11b BV421 1:400 (M1/70, BioLegend), CD11b PE-Cy-7 1:400 (M1/70, eBioscience), CD11c BV421 1:100 (N418, BioLegend), CD16/32 unconjugated 10 μg ml⁻¹ (93, BioLegend), CD19 BV421 (6D5, BioLegend), CD19 APC 1:400 (1D3, BD Pharmingen), CD45 BV421 1:400 (30-F11, BioLegend), CD45 BV785 1:400 (30-F11, BioLegend), CD49b APC 1:100 (DX5, BD Pharmingen), CD90.2 APC-Cy7 1:400 (30-H12, BioLegend), CD117 PE 1:800 (2B8, eBioscience), CD117 APC 1:800 (2B8, BD Pharmingen), CD117 BV711 1:800 (2B8, BioLegend), FcεRI APC 1:200 (MAR-1, eBioscience), Gr-1 BV421 1:800 (RB6-8C5, BioLegend), Gr-1 BV605 1:200 (RB6-8C5, BioLegend), IgE PE 1:100 (RME1, BioLegend), IgE BV786 1:100 (RME-1, BD Pharmingen), IgE BV421 1:100 (RME-1, BD Pharmingen), Ly6G PerCP-Cy5.5 1:100 (1A8, BD Pharmingen), MHCII A700 1:100 (M5/114.15.2, eBioscience), Siglec-F BV421 1:100 (E50-2440, BD Pharmingen), Siglec-F PE 1:100 (E50-2440, BD Pharmingen), Ter119 BV421 1:200 (Ter119, BioLegend), 5-HT unconjugated 0.11 μg ml⁻¹ (5HT-H209, Dako) and mouse-IgG1 PE 1:100 (RMG1-1, BioLegend).