Isotype matched control mouse igg2a mab

As previouslty described^{20 (link)}, intracellular WASp expression in Peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry analysis. After fixation and perforation, 2 × 10⁶ PBMCs in each tube were incubated with 0.25 mg/ml purified mouse anti-human WASp mAb (BD Pharmingen) or isotype-matched control mouse IgG2a mAb (BD Pharmingen) at 4 °C for 30 min. Then all the cells were incubated with 1:50 diluted PE-conjugated Rat anti-mouse IgG2a (BioLegend) and reacted at 4 °C for 30 min. The PE- labeled cells was assessed by flow cytometry.

Intracellular detection with anti-WASp monoclonal antibody was performed as described before (21 (link)). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Axis-Shield, Norway) density gradient centrifugation and washed twice with phosphate-buffered saline (PBS). Cytofix/Cytoperm solution from CytoStain Kits (Pharmingen, San Diego, CA) was added to suspended 2 × 10⁶ PBMCs in each tube at 4°C for 20 min. After washing twice in Perm/Wash solution, they were incubated with 0.25 mg/ml purified mouse anti-human WASp mAb (BD Pharmingen) or isotype-matched control mouse IgG2a mAb (BD Pharmingen) at 4°C for 30 min and washed twice. Subsequently, phycoerythrin (PE)-conjugated goat anti-mouse IgG2a (BD Pharmingen) was added to all cells and reacted at 4°C for 30 min. WASp expression in PBMCs was analyzed by FACS Calibur, using CellQuest software (Becton Dickinson).