Smrnp

ELISpot assays were performed as previously described (5 (link), 6 (link)). Briefly, splenocytes in RPMI containing 10% fetal bovine serum were plated at a concentration of 1 x 10⁶ cells/well onto salmon sperm dsDNA- (Invitrogen), nucleosome- (histone from Sigma Aldrich plated on a layer of dsDNA coating)- or SmRNP- (Arotec Diagnostics) coated multiscreen 96-well filtration plates (Millipore, Bedford, MA). Serially diluted (1:2) cells were incubated for 12 h at 37°C. dsDNA-, nucleosome-, and SmRNP-specific AFCs were detected by biotinylated anti-kappa Ab (Invitrogen) and streptavidin (SA)-alkaline phosphatase (Vector Laboratories) or alkaline phosphatase-conjugated anti-mouse IgG (Molecular Probes). Plates were developed using the Vector Blue Alkaline phosphatase Substrate Kit III (Vector Laboratories). ELISpots were enumerated and analyzed using a computerized ELISpot plate imaging/ analysis system (Cellular Technology).

ELISpot assays were performed as previously described (25 (link), 43 (link)). Briefly, splenocytes in RPMI containing 10% fetal bovine serum were plated at a concentration of 1 × 10⁶ cells/well onto salmon sperm dsDNA- (Invitrogen), nucleosome- (histone from Sigma Aldrich plated on a layer of dsDNA coating)- or SmRNP- (Arotec Diagnostics) coated multiscreen 96-well filtration plates (Millipore, Bedford, MA). Serially diluted (1:2) cells were incubated for 12 h at 37°C. dsDNA-, nucleosome-, and SmRNP-specific AFCs were detected by biotinylated anti-kappa Ab (Invitrogen) and streptavidin (SA)-alkaline phosphatase (Vector Laboratories) or alkaline phosphatase-conjugated anti-mouse IgG (Molecular Probes). Plates were developed using the Vector Blue Alkaline phosphatase Substrate Kit III (Vector Laboratories). ELISpots were enumerated and analyzed using a computerized ELISpot plate imaging/ analysis system (Cellular Technology).