Srbcs

For SRBC immunization, 1 ml sterile SRBCs (catalog no. 12977755, Thermo Fisher Scientific) were washed twice with 10 ml ice-cold PBS and reconstituted in 3 ml PBS and administered as 0.2-ml injections intraperitoneally. In some experiments, an enhanced SRBC immunization method was followed to maximize GC B cell yield by immunizing mice with 0.1 ml SRBC on day 0 followed by a 0.2-ml second injection on day 5 (ref. ^{53 (link)}). For protein antigen immunizations, 50 μg NP_(30–39)-CGG (catalog no. N-5055D-5, Biosearch Tech) was mixed with alum (Thermo Fisher Scientific) or PBS for boost immunization at a 1:1 ratio and rotated at 20 °C (room temperature) for 30 min before intraperitoneal injection. For NP-CGG- and SRBC-based immunizations, day 14 and day 12 were used as read-out time points, respectively, unless specified otherwise.

To chemically link HEL to SRBCs, HEL was first activated with Sulfo-NHS (Thermofisher Scientific) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in MES buffer for 15 minutes at room temperature. Activated HEL was passed over a Zeba spin desalting column equilibrated with PBS at pH 7.4, lyophilized and stored at -20°C until use. SRBCs (Rockland Laboratories) were washed in 1x DPBS and resuspended in 1x DPBS at a 2 to 1 ratio. Activated HEL was quickly thawed at room temperature and reconstituted in deionized water. Reconstituted activated HEL was immediately added at an equal volume to the packed SRBCs and incubated for 25 minutes at room temperature. The cells were washed in 1x DPBS. To confirm HEL linkage to SRBCs, HEL SRBCs and unlabeled SRBCs were stained with or without anti-HEL antibodies (clone: 2F4 and 4B7; Bioxcell) for 15 minutes at room temperature. Cells were washed and then resuspended in APC anti-mouse IgG for 15 minutes at room temperature. Samples were run on a 4 laser Cytek Aurora and analyzed using FlowJo software. B6 and S129/B6 FVIII deficient mice were transfused via the retro-orbital plexus with 50 μl packed HEL SRBCs diluted to a 150 μl total volume in 1x DPBS.