Splenic B cell Ca2+ flux assay

Splenic B cells were freshly isolated by density gradient centrifugation and stained as previously described (16 (link)). In brief, cells were stained with CD19-FITC and CD5-APC mAb for 20 min, washed and loaded with 10 μg/ml FuraRed and 0.02% Pluronic F127 (both from Thermo Fisher) for 25 min at 30°C. Baseline was recorded in 4 mM Ca²⁺ Krebs-Ringer solution for 30 s and Ca²⁺ mobilization was assessed after stimulation of the cells with 10 μg/mL α-IgM F(ab′)₂ (Jackson ImmunoResearch). After 3 min of recording, 1 μM Ionomycin was added as a positive control. Increases in free intracellular Ca²⁺ were measured in real-time with the Canto II cytometer. To determine the Ca²⁺ flux, the ratio of bound and unbound FuraRed was calculated with the FlowJo software.