Goat anti mouse igg ap

ELISpot plates (Millipore, Ref# MSIPS4W10; Multiscreen HTS) were coated with 0.01% poly-L lysine for 1h at RT, followed by coating with 100 μg/ ml calf thymus DNA (Sigma Aldrich) or 10 μg/ ml calf thymus nucleosomes (Arotec diagnostics) overnight at 4°C. Blocked with PBS + 5% FCS for 2–3h at RT. Freshly isolated splenocytes or BM cells were resuspended in freshly prepared warm 15% RPMI 1640 media + antibiotics + 1mM L-glutamine at 20 × 10⁶ cells/ ml. 1×10⁶ total cells were plated on the top wells and serially double diluted (1:2). ELISpot plates were then incubated for 18h at 37°C with 5% CO₂. Washed with PBS + 1% FCS to remove the cells and probed with a 1:500 dilution of goat anti-mouse IgG AP (Jackson Immunoresearch) for 3–4h at 4°C. After washing, plates were developed using the Vector Blue AP Substrate Kit III (Vector Labs). Spots were captured and counted using ImmunoSpot S6 Analyzer (Cellular Technology Limited, Shaker Heights, OH). For analysis of total IgG-secreting AFCs, ELISPot plates were coated with goat anti-mouse IgG (SouthernBiotech) at 5μg/ml and blocked with PBS + 5% BSA. Plates were washed with PBS + 1% BSA and probed with a 1:500 dilution of goat anti-mouse IgG AP (Jackson Immunoresearch).