Biotin anti ly5.1 bp 1 6c3 fitc anti cd23 b3b4 pe cy5 streptavidin sa

The following antibodies were utilized for flow cytometric analysis of mouse splenocytes or bone marrow cells: PacBlue-anti-B220 (RA3-6B2); Alexa Fluor 700-anti-CD4 (RM4-5); PE-anti-PD-1 (29F.1A12); PerCP-Cy5.5-anti-CD69 (H1.2F3); APC-Cy7- anti-CD25 (PC61); Cy5-anti-CD86 (GL1); PeCy7-anti-CD95 (FAS, Jo2); PeCy7-anti-MHC-II (M5/114.15.2); APC-anti-CD24 (HSA) (M1/69); Biotin-anti-Ly5.1 (BP-1) (6C3); FITC-anti-CD23 (B3B4); PE-Cy5-streptavidin (SA) were from purchased from BioLegend, San Diego, CA. Biotin-anti-CXCR5 (2G8) from BD Pharmingen, San Diego, CA. FITC-peanutagglutinin (PNA) from Vector Labs, Burlingame, CA. PE-anti-IgM (eB121-15F9); APC anti-CD93 (AA4.1); FITC-anti-F4/80 (BM8) from eBiosciences, San Diego, CA. The following antibodies were utilized for phosphoflow analysis of purified mouse B cells: PE-anti-Btk (pY223)/ItK (pY180) (N35-86); Alexa Fluor 647-anti-ERK1/2 (pT202/pY204) (20A); Pacific Blue-anti-p38 MAPK (pT180/pY182); Alexa Fluor 488-anti-Syk (pY348) (I120-722). Stained cells were analyzed using the BD LSR II flow cytometer (BD Biosciences, Franklin lakes, NJ). Data were acquired using FACSDiva software (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, San Carlos, CA).

The following antibodies were utilized for flow cytometric analysis of mouse splenocytes or bone marrow cells: PacBlue-anti-B220 (RA3-6B2); Alexa Fluor 700-anti-CD4 (RM4-5); PE-anti-PD-1 (29F.1A12); PerCP-Cy5.5-anti-CD69 (H1.2F3); APC-anti-TCR Vα2 (B20.1); APC-Cy7- anti-CD25 (PC61); Cy5-anti-CD86 (GL1); PeCy7-anti-CD95 (FAS, Jo2); PeCy7-anti-MHC-II (M5/114.15.2); APC-anti-CD24 (HSA) (M1/69); Biotin-anti-Ly5.1 (BP-1) (6C3); FITC-anti-CD23 (B3B4); PE-Cy5-streptavidin (SA) were from purchased from BioLegend, San Diego, CA. Biotin-anti-CXCR5 (2G8); FITC-anti-CD11c (HL3); FITC-anti-CD43 (S7) from BD Pharmingen, San Diego, CA. FITC-peanut-agglutinin (PNA) from Vector Labs, Burlingame, CA. PE-anti-IgM (eB121-15F9); APC-anti CD93 (AA4.1); FITC-anti-F4/80 (BM8) from eBiosciences, San Diego, CA. Stained cells were analyzed using the BD LSR II flow cytometer (BD Biosciences, Franklin lakes, NJ). Data were acquired using FACSDiva software (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, San Carlos, CA). Dead cells were quantified by flow cytometry using 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO).