Igd apc cy7 clone 11 26c2a

Spleens were collected and splenocytes were isolated in a single cell suspension after being passed through a 100 micron filter. Splenocytes were then subjected to red blood cell lysis by treatment with red blood cell lysis buffer (Sigma # R7757) per manufacturer’s recommendation. Peritoneal cells were collected with 10 mLs of cold 1% FBS DMEM. β-lactamase activity was detected using the LiveBLAzer FRET-BG/Loading Kit with CCF2-AM (ThermoFischer Scientific # K1025) as previously described [17 (link),29 (link),32 (link)]. Cell surface antibodies used were CD19-AlexaFluor 700 (clone eBio1D3, eBioscience # 56-0193-81), CD38-APC (clone 90,eBiosciences #17-0382-81), IgD-APC-Cy7 (clone 11-26c.2a, Biolegend # 405716), and CD5-APC (clone 53–7.3, eBioscience # 17-0051-81). Fc blocking antibody 24G2 was used in staining to prevent antibody binding to cellular Fc receptors.

CD3-FITC clone 145–2C11, CD4-PE-Cy7 clone RM4–5, CD4-PE clone RM4–4, CD11a-FITC clone M17/4, CD49d-PE clone R1–2, CXCR5-biotin clone L138D7, PE-Cy7 streptavidin, CD44-FITC and CD44-APC-Cy7 clone IM7, PD-1 APC clone 29F.1A12, GL7-PacBlue and GL7-PE clone GL7, CD19-PerCP-Cy5.5 clone 6B2, CD138-BV421 clone 281–2, IgD-APC-Cy7 clone 11–26c2a, CD95-PE clone SA367H8, CD73-BV605 clone TY/11.8, CD71-BV421 Clone RI7217, and CD80-PE clone 90, were purchased from Biolegend (San Diego, CA). BcL6-PE-CF594 clone K112–91 was purchased from BD Biosciences, San Jose, CA)