B220 apc clone ra3 6b2

Cells were stained for flow cytometry at 4° C for 30 minutes in FACS buffer (PBS, 1% BSA, and 1mM EDTA) plus appropriate antibodies and fixed in 0.1% paraformaldehyde for at least 1 hour. Magnetically sorted B cells were gated on CD19+ B220+ CD11b− and CD11c− events. Magnetically sorted CD4 T cells were gated on CD3+ CD8− CD4+ CD11b− and CD11c− events. Primary BMDMs were gated on CD11b+ CD11c− MHCII+ cells. RAW 264.7 macrophages, primary BMDMs, T cells, and B cells were also stained with anti-PD-1 antibody for analysis. Antibodies used were CD4 PerCP-Cy5.5 (clone RM45, Tonbo Biosciences Corp., San Diego, CA), CD8 FITC (clone 53-6.7, Tonbo Biosciences Corp., San Diego, CA), CD19 FITC (clone 1D3, BD Pharmingen, San Diego, CA), B220 APC (clone RA3-6B2, BD Pharmingen, San Diego, CA), CD11b FITC (clone M1/70 BD Pharmingen, San Diego, CA), CD11C APC (clone HL3, BD Pharmingen, San Diego, CA), PD-1 PE (clone RMP1-30, Biolegend Inc., San Diego, CA). Flow cytometry was performed on a BD LSR-II and analyzed using FlowJo 9.6.4 software.

Viability dye (cat. L34968) and CFSE (cat. 65-0850-84) were both purchased from Invitrogen. The following monoclonal antibodies (mAbs) were also purchased from Invitrogen: H-2Kd APC (clone SF1-1.1.1, cat. 17-5957-82), CD8a PE-Cy7 (clone 53-6.7, cat. 25-0081-82), MHCII Pacific Blue (clone M5/114.15.2, cat. 48-5321-8), and CD11c PE-Cy7 (clone N418, cat. 12-7177-82). The following mAbs were purchased from Biolegend: CD4 APC/cy7 (clone GK1.5, cat. 100414), CD11b APC (clone M1/70, cat. 101212), CD86 PECy7 (clone GL-1, cat. 105014). B220 APC (clone RA3-6B2) was purchased from BD Pharmingen (cat. 553092). We obtained anti-CD5.1 (Ly5.1)-FITC, clone H11-86.1, from BD Pharmingen (cat 557487).