A20 cell line, derived from mouse B cell lymphoma on BALB/c background [13 (link)], was obtained from American Type Culture Collection (Manassas, VA). Primary cells were isolated from bone marrow (BM), spleen (SPL) or peripheral blood (PB) of B6 by standard methods. The A20 transfectants or primary cells were stained with the following antibodies (Abs): anti-mouse CEACAM1 mAb, CC1 (kindly provided by Dr. Kathlyn Holmes, University of Colorado, CO), anti-IgG1, FITC-anti-B220, PE-anti-IgM, FITC-anti-CD4, PE-anti-CD3ε, PerCP-anti-B220, PE-anti-CD43, PE-anti-CD25, PE-anti-Igκ, PE-anti-CD138, PE-anti-CD5, PE-anti-H-2Kd, PE-anti-I-Ad, PE-anti-CD69, PE-anti-CD80 and PE-anti-CD86 Abs (BD Biosciences, San Jose, CA). Data acquisition was performed using FACS Calibur™ and analysis software Cell Quest™ (BD Biosciences).
In some experiments, the A20 transfectants were treated with Fluo-4® (Dojindo, Kumamoto, Japan), and applied for FACS to analyze intracellular Ca2+ influx. During the acquisition, cells were treated with anti-Igκ (Southern Biotech, Birmingham, AL) to stimulate BCR signaling, and kinetics for Ca2+ influx were measured at FL2.
In some experiments, the A20 transfectants were treated with Fluo-4® (Dojindo, Kumamoto, Japan), and applied for FACS to analyze intracellular Ca2+ influx. During the acquisition, cells were treated with anti-Igκ (Southern Biotech, Birmingham, AL) to stimulate BCR signaling, and kinetics for Ca2+ influx were measured at FL2.