Anti gl7 fitc

The Peyer’s patches were excised from 8-week-old ROD1 WT or KO mice and then gently dissociated by passage through 70 μm cell strainers. Germinal center B cells were double stained by anti-GL7-FITC (Biolegend, Catalog # 144603) and anti-Fas-APC (Biolegend, Catalog # 152603) antibodies, and were directly sorted into mouse genomic DNA extraction buffer by FACS Jazz (BD Biosciences). Genomic DNA was then purified following manufacturer’s protocol (Selleck, Catalog # B40013). JH4 intron was amplified by PCR using KOD DNA polymerase and a reported primer pair (F: 5′-GCCTGACATCTGAGGACTCTGC-3′ and R: 5′-CCTCTCCAGTTTCGGCTGAATCC-3′).^{32 (link)} JH4 amplicons were inserted into pCR-Blunt II-TOPO (Life technologies, Catalog # K280002) and sequenced with M13 primers by BGI. The mutation frequency was calculated from successfully sequenced clones by counting overall C/G or G/C mutations to total sequenced bases.

Spleens and inguinal lymph nodes were collected and single-cell suspensions were generated in IMDM 10% FCS Pen-Strep. Cells were surface stained to quantify cell populations. T cell panel: anti-CD3 Alexa 700 (BioLegend; clone 17A2), anti-CD4 PE-Cy7 (BioLegend; clone RM4-5), anti-CD8 V500 (BD Horizon, clone 53-6.7), anti-CD62L APC (BD Pharmingen; clone MEL-14), anti-CD44 Pacific Blue (clone IM7, grown in-house), anti-CXCR5 PE (BioLegend; clone J252D4), and anti-CCR7 PerCP (BioLegend; clone 4B12). B cell panel: anti-B220 PE (BioLegend; clone RA3-6B2), anti-CD38 APC (BioLegend; clone 90), anti-Fas PE-Cy7 (BioLegend; clone Jo2), anti-GL7 FITC (BioLegend; clone GL7), anti-MHCII Pacific Blue (BioLegend; clone M5/114.15.2), anti-CD19 Alexa 700 (BioLegend; clone 6D5), and anti-CD138 Percp (BioLegend; clone 281-2). CD4⁺ T_CM were CD3⁺CD4⁺CD44^highCD62^high, CD4⁺ T_EM were CD3⁺CD4⁺CD44^highCD62L^low, T follicular helper cells (T_FH) were CD3⁺CD4⁺CXCR5⁺, germinal center (GC) B cells were B220⁺CD19⁺GL7⁺Fas⁺, and plasma cells B220⁺CD19⁺CD138⁺CD38⁺.