α igm

Splenic B cells were stained with CD19-BV510 and CD5-APC mAb for 15 min, washed and diluted in FCS. Cells were stimulated with 10 μg/mL α-IgM (Southern Biotech) for the indicated time points at 37°C. Cells were immediately fixed and permeabilized with the PerFix EXPOSE Kit (Beckman Coulter) according to the manufacturer's instructions. Intracellular staining of ERK1/2/P-ERK1/2(T202/Y204) (1:400/1:800) and SYK/P-SYK(Y525/526) (both 1:200) and respective isotype controls was performed, followed by detection with an α-rabbit F(ab′)₂-PE conjugate (1:250) (all from Cell Signaling Technology) and measured with the LSRFortessa cytometer (BD Bioscience).

PBMCs of CLL patients were freshly isolated by ficoll gradient centrifugation were incubated with CD19-APC-Cy7 (HIB19, 1:100) (eBioscience) for 20 min. Cells were washed with PBS w/o Mg²⁺/Ca²⁺ und incubated in 250 µL RPMI 1640 (Invitrogen.) containing 5% FCS (Biochrom), 10 µg/ml FuraRed (Invitrogen) and 0.02% Pluronic F127 (Invitrogen) for 25 min at 30 °C. Cell suspensions were subsequently diluted with 250 µl of medium containing 10% FCS and incubated at 37 °C for another 10 min. Cells were then immediately diluted with 1 ml 0 mM Ca²⁺ Krebs-Ringer solution containing 10 mM Hepes, pH 7.0, 140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose and 0.5 mM EGTA and washed with 1 mL 0 mM Ca²⁺ Krebs-Ringer solution. For the measurements, the cells were diluted with 200 µL 4 mM Ca²⁺ Krebs-Ringer solution and a baseline was recorded for 60 s. Ca²⁺ movement was assessed after stimulation of the cells with 20 µg/ml α-IgM (Southern Biotech). After 4 min of recording, 1 µM Ionomycin was added as a positive control. Increases in free intracellular Ca²⁺ were measured in real-time on a Canto II (BD). To determine the Ca²⁺ flux, the ratio of bound and unbound FuraRed was calculated with FlowJo 8.5.3 software. The gating strategy used for the Ca²⁺ measurements is provided in Supplementary Fig. 11.