Quantification of Antibody-Secreting Cells by ELISpot

ELISpot assays were performed as previously described (16 (link)). Briefly, splenocytes in 10% RPMI were plated at a concentration of 1 × 10⁵ cells/well onto anti-IgM or anti-IgG (Invitrogen, Grand Island, NY) coated, or at 1 × 10⁶ cells/well on dsDNA-, histone-, or nucleosome-coated multiscreen 96-well filtration plates (Millipore, Bedford, MA). Serially diluted (1:2) cells were incubated for 6 h at 37°C. IgM-producing AFCs were detected using biotinylated anti-mouse IgM (Jackson Immunoresearch, West Grove, PA) and streptavidin (SA)-alkaline phosphatase (Vector Laboratories, Burlingame, CA). IgG-producing AFCs were detected using alkaline phosphatase-conjugated anti-mouse IgG (Molecular Probes, Grand Island, NY). dsDNA-, histone-, and nucleosome-specific AFCs were detected by biotinylated anti-kappa Ab (Invitrogen, Grand Island, NY) and streptavidin (SA)-alkaline phosphatase (Vector Laboratories, Burlingame, CA) or alkaline phosphatase-conjugated anti-mouse IgG (Molecular Probes, Grand Island, NY). Plates were developed using the Vector Blue Alkaline phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA). ELISpots were enumerated and analyzed using a computerized imaging/analysis system (Cellular Technology, Shaker Heights, OH).