Pe anti igκ

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-2K^d, PE-anti-I-A^d, 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.

Spleens were homogenized by filtering through a 70-μm cell strainer and red blood cells were lysed with ACK buffer (Thermo Scientific). Resting T and B cells were purified by negative magnetic-activated cell sorting (MACS) using the mouse CD4⁺ T cell Isolation Kit and anti-CD43 Microbeads (Miltenyi Biotec), respectively, according to the manufacturer’s protocol. For all imaging experiments except intravital imaging of T–B border interactions, NP-binding B cells were quantified by flow cytometry using NP-PE (ThermoFisher), and total B cells containing the specified number of NP-binding B cells were transferred. When imaging T–B border interactions, we enriched for Igλ⁺ B cells by incubating splenocytes with anti-Igκ PE (Clone 187.1, BD Biosciences) at 0.7 μg/ml for 30 min prior to MACS using anti-CD43 and anti-PE Microbeads combined (Miltenyi Biotec). For transfers of resting CD4⁺ Foxp3^– cells, negative MACS isolation of CD4⁺ T cells was followed by FACS of unstained GFP^– dsRed⁺ cells prior to adoptive transfer. For characterization of the Rosa26^Foxp3 mouse, CD4⁺ T cells were enriched using the Dynabeads Untouched Mouse CD4 Cells Kit (Life Technologies 11415D). A total of 1.5×10⁷ enriched CD45.2⁺ CD4 T cells from Foxp3^RFPRosa26^Foxp3 mice positive or negative for the CD4-CreERT2 transgene were transferred retro-orbitally into CD45.1⁺ recipients.