Indo 1 am

Spleen tissue from BTK^C481S and BTK^WT mice were passed through a 70-μm filter into complete RPMI media supplemented with FBS (2% v/v) and 1M HEPES (1% v/v). Erythrocytes were lysed by resuspension in 1–2 mLs of ACK buffer. B cell suspensions were purified by negative selection using MACS CD43 beads, quantified, and mixed at equal concentrations. B cells were resuspended to 10⁷ cells/mL in PBE with 1x PowerLoad Concentrate (Thermo Fisher Scientific) and Indo-1 AM [2 μM] (Thermo Fisher Scientific). Cells were incubated, protected from light, at 37C for 30 minutes. After loading, cells were washed 2x and 2×10⁶ cells were plated in a 96 well plate with ibrutinib (concentrations indicated) for 30 minutes at 37C. Cells were washed 2x with RPMI 1640 medium, no phenol red (Thermo Fisher Scientific), 1% BSA, and rested in RPMI buffer on ice with surface-staining antibodies for 30 minutes. Stimulation was performed by addition of biotinylated Goat-Anti-mouse IgM [20 μg/mL] followed by Streptavidin (Jackson ImmunoResearch) [40 μg/mL].

After 2 washes with serum-free RPMI medium, cells were incubated with serum-free RPMI with 5 µM Indo-1 AM (Thermo-Fisher, I1203) for 30 minutes. Cells were then washed 3 times with PBS. Flow cytometry was performed by ratiometric analysis of cytoplasmic calcium-bound vs unbound Indo-1 vs time. Analysis was performed for 30 seconds on untreated cells prior to the addition of treatment agents.

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.

Five million cells were centrifuged for 5 min at 300 g and the medium was discarded. The cell pellet was resuspended in 1 ml stimulation medium (RPMI 1640 medium supplemented with 1% FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 µg/ml streptomycin) with 0.1% (v/v) pluronic F-127 and 4 µM Indo-1 AM (all Thermo Fisher) and incubated in the dark for 30 min at 37°C. The stained cells were washed and kept on ice in the dark until the measurement. For calcium influx, cells were diluted 1:20 with pre-warmed stimulation medium and maintained at 37°C during the event collection on a MACSQuant X flow cytometer. After fluorescence baseline acquisition, stimuli were added or activated by illumination as depicted. If not indicated otherwise PhyBt were added to a final concentration of 20 nM.
For the graphs showing the percent of responding cells, the events above the 90^th percentile during baseline acquisition were quantified using FlowJo 9 (FlowJo LLC). To calculate the calcium influx values (a.u.), average Indo-1 ratio values after stimuli addition (250–400 s) minus baseline values (30–60 s) were normalized for each experiment using an internal control of 20 nM PhyBt(660) in the dark.

As described^{63 (link)}, thymocytes from WT mice were labeled with CFSE (20 nM) in PBS for 10’ at 37 °C, washed and mixed with thymocytes from SKG mice(CSFE⁻) at 1:1 ratio. Cells were then loaded with Indo-1 AM (ThermoFisher) 2 uM with 4 μM probenecid (ThermoFisher) in RPMI with 1%FBS. After washing, cells were stained with antibodies against CD4 and CD8α as well as biotinylated anti-CD3ε antibodies (10 μg/ml, 145-2C11; Biolegend) on ice. The cells were stimulated by streptavidin crosslinking in PBS and calcium flux was initiated after addition of CaCl₂(5 mM). The mean fluorescence ratio of Indo-1 violet to Indo-1 blue was calculated using FlowJo software (Tree Star, Inc).