F ab 2 rabbit anti mouse igg

Calcium influx into total T lymphocytes from freshly thawed PBMCs was analyzed by real-time flow cytometry, as previously described (Hauck et al., 2012 (link)). Briefly, cells were loaded with indo-1 acetoxymethyl ester (Molecular Probes, 5 μM) in the presence of 2.5 mM probenecid (PowerLoad; Molecular Probes), washed, surface-stained for CD4 and CD8, washed, stimulated with anti-CD3 mAb (Clone: OKT3, 1 μg/ml) followed by F(ab′)₂ rabbit–anti-mouse IgG (Jackson Immunoresearch, 10 μg/ml), and then further stimulated with ionomycin (Sigma-Aldrich, 1 μM). Cells were analyzed with a FACSAria flow cytometer (BD Biosciences). Intracellular Ca²⁺ levels were quantified by calculating the normalized ratio of fluorescence intensity (Ca²⁺-bound to Ca²⁺-free Indo-1) and plotted as a function of time.

For analyzing BCR-induced phosphorylation and ubiquitylation signaling, A20-, A20.2J-, and A20.2J-derived cell lines were serum-starved for 4 h. BCRs were stimulated with 20 μg/ml of the F(ab′)2 of rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories). For the isolation of BCR signalosomes, “medium” and “heavy” labeled cells were stimulated with biotinylated F(ab′)2 rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories), and unstimulated (without anti-mouse IgG) “light” labeled cells were used as control. After stimulation, cold phosphate-buffered saline (PBS) was immediately added to the cell suspension to stop the stimulation and cells were harvested by centrifugation, washed twice with PBS, and were either frozen in liquid nitrogen or directly processed for subsequent experiments.