Immunization and Enzyme-linked immunosorbent assay (ELISA)
WT and CCR2 KO mice were injected intraperitoneally (i.p.) with T cell dependent antigen 4-hydroxy-3-nitrophenylacetyl conjugated keyhole limpet hemo-cyanin (NP-KLH, 40 µg/mouse) (N-5060-25, Biosearch Technologies), supplemented with Freund’s incomplete adjuvant (FIA) (S6322-1VL, Sigma Aldrich). Fourteen days post-prime, 100 μl of blood was taken from the tail vein, then mice were boosted with the same reagents. Five days later, the mice were euthanized and the spleens were harvested. FCM was performed to test the ratio and absolute cell number of B cell subsets. ELISA was performed using NP30-bovine serum albumin (BSA)-coated the ELISA kit (Bethyl Laboratories) was perform as indicated in the manufacturer’s information to determine the titer of the NP-specific IgM and IgG1. Serum anti-dsDNA antibody was also quantified by ELISA as previously described.24