Facscanto cytometer

Flow cytometry was used to assess the following cell lineages: B cells (CD3⁻CD19⁺), plasma cells (CD3⁻CD138⁺), FCER2⁺ B cells, and FCER2⁻ B cells. PBMCs from the above patients were isolated from whole blood using density gradient centrifugation in Ficoll/Hypaque for 20 minutes at 400 × g with the brake-off. The PBMCs were washed and stained with the indicated fluorescence-labeled antihuman antibodies for 30 min on ice and protected from light. The cells were then washed and fixed in 2% formaldehyde. The samples were acquired using a FACSCanto cytometer and analyzed using FlowJo v.10 (Treestar, Seattle, California, USA). The corresponding isotype controls were used to determine the negative gate. Antibodies used in this study included CD23-FITC (#338505), CD19-PE (#302207), CD3-APC (#300311), and CD138-APC-Fire^TM 750 (#352315, BioLegend, San Diego, California, USA).

Splenic B cells pretreated or not with CX-4945 (Sellekchem) 5μM for 3 hours, were loaded with Fluo4-AM (4μg/ml; Molecular Probes) and FuraRed (16μg/ml; Molecular Probes), sulfinpyrazone (250μM; Sigma) and Pluronic F-127 (0,1% w/v, Life Technologies) in RPMI for 30 min at 37°C in the dark. Cells were washed with RPMI and resuspended in warm PBS at the density of 10⁶ cells/ml. After recording for 30 sec to establish the baseline, cells were stimulated with 20 μg/ml F(ab’)₂ rabbit anti–mouse IgM (Jackson ImmunoResearch Laboratories, Inc.) for 6.5 min. Then, ionomycin (2 μg/ml) was added to allow the complete emptying of Ca⁺⁺ stores. Measurements were monitored using a FACSCanto cytometer and analyzed using FlowJo software (Tree Star).