Isolation and Sorting of Germinal Center B Cells

GC B cells were generated and sorted using a previously described protocol (Hwang et al., 2015 (link)). Briefly, 6–12-week-old M2A-GFP KI mice were immunized with sheep’s red blood cells. After 8–10 days, total B cells from the spleens and lymph nodes were isolated using the Negative Selection B cell isolation kit (StemCell Technologies) according to the manufacturer’s instructions. Dead cells were stained using Zombie Yellow viability stain (BioLegend) and Fc receptors were blocked with the mouse TruStain FcX antibody (#156604). Cells were immunostained with anti-mouse CD38 (#102719), B220 (#103235), and GL-7 (#144617) purchased from BioLegend. GC B cells were sorted on a BD Aria III FACs sorter (Beckton Dickinson) for GFP+, Zombie Yellow^-, B220⁺, CD38^low and GL-7⁺ cells, and were used immediately.