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