SRBC immunization, plasma cell enumeration and anti-SRBC antibody tittering

Mice were immunized with sterile citrated SRBC (Cedarlane, CL2580). One milliliter of the SRBC suspension was washed twice with 50 mL of PBS, and the resulting pellet was resuspended in 3.6 mL of PBS. Each animal was immunized i.p. with 0.1 mL of this suspension. Twelve days after immunization, the animals were euthanized, and their spleens and cardiac blood samples were collected. Single-cell splenocyte suspensions were prepared, hemolyzed, filtered and counted using a MoxiFlow cytometer. For plasma cell staining, samples were stored at 4 °C in EasySep buffer or FACS buffer (containing FBS) prior to staining. Then, the samples were incubated with 1:50 Fc block (anti-CD16/CD32) for 10 min at room temperature before washing with 1 mL EasySep or FACS buffer and addition of the antibody cocktail (anti-CD138-BV421, anti-CD267/TACI-PE, anti-CD4-FITC, anti-B220-PerCP, and FVS-780 viability dye (1:100 dilution of each antibody and 1:1000 dilution of viability dye in a total volume of 50 µl per sample, for 30 min at 37 °C). Plasma cells (CD138⁺/CD267⁺) were enumerated upon gating on the lymphocyte subset.

Serial dilutions of serum in PBS (from 1:2 to 1:4096; 0.1 mL per well) were incubated with 0.1 mL of the SRBC suspension diluted to 1% in PBS for 30 min at 37 °C. Titers were calculated after overnight storage of the plates at 4 °C as the dilution of serum falling in the middle of the zone of equivalency (the wells with a positive agglutination reaction but no settling of SRBC at the bottom).