Perm buffer 2

The expression of BTK in monocytes was evaluated by immunostaining from whole blood as previously described (7 (link), 8 (link)). In brief, the red blood cells were removed using Lyse/Fix Buffer (BD Pharmingen, San Diego, CA), then incubated with CD14–PE (clone Mϕ, BD Pharmingen) for 20 min, permeabilized using Perm Buffer II (BD Pharmingen), and then stained with AlexaFluor647-conjugated anti-BTK Ab (clone53/BTK, BD Pharmingen) or isotype IgG2a (clone MOPC-173, BD Pharmingen) to identify epitopes between 2 and 172 amino acids of the Pleckstrin homology (PH) and Tec homology (TH) domains within the gating monocytes. The threshold of FSC was set as 5,000 and the data was analyzed by FlowJo 7.6 (Treestar, USA).

PBMCs were rested for 30 min in RPMI at 37°C after isolation and stimulated with 19.5 µg/ml aBCR F(ab)₂ IgM/IgA/IgG or RPMI for 5 min. Cells were then lysed/fixed (BD Lyse/Fix Buffer, BD Biosciences) and subsequently permeabilized with Perm Buffer II (BD Biosciences) according to the manufacturer’s instructions. Intracellular staining of pSYK Y³⁵² and markers to identify respective subsets was carried out for 1 h at room temperature and cells were acquired on a BD LSR Fortessa X-20.

Stimulated T cells and IL-12 receptor-specific siRNA-transfected T cells were stained with anti-mouse CD4-peridinin-chlorophyll protein and anti-mouse CD25-allophycocyanin for 30 min at 4 °C. The cells were permeabilized and fixed with CytoPerm/CytoFix (BD Biosciences, San Diego, CA, USA) in accordance with the manufacturer’s protocol; further stained with anti-mouse Foxp3-phycoerythrin (PE) and/or anti-mouse IL-17-fluorescein isothiocyanate (FITC), anti-mouse IFN-γ-PE, and anti-mouse IL-4-PE (all from eBioscience, San Diego, CA, USA); and subjected to flow cytometric analysis (FACSCalibur; BD Biosciences). For p-STAT3 Y705 and S727 and p-STAT5 analysis, splenocytes were treated with p40-EBI3 (1 and 10 μg/mL) and IL-6 for 1 h; the cells were then stained with anti-mouse CD4-FITC. The cells were fixed and permeabilized with Lyse/Fix Buffer (BD Pharmingen, San Jose, CA, USA) and Perm Buffer II (BD Pharmingen). The permeabilized cells were stained with anti-p-STAT3 Y705-PE, anti-p-STAT3-S727-PE, or anti-p-STAT5-PE (BD Pharmingen) and subjected to flow cytometric analysis (CytoFLEX; Beckman Coulter, Fullerton, CA, USA).