ELISPOT Assay for Anti-dsDNA Plasma Cells

ELISPOT for dsDNA producing plasma cells was performed as described (33 (link)). Briefly, ELISPOT MultiScreen plates (Millipore, cat. MSIPS4W10) were pre-coated with 10μg/mL methylated BSA (Sigma) at 37 C for 2h followed by 10μg/mL calf thymus double stranded DNA (Sigma) or PBS (control group) at 4 C overnight. Femurs and spleen were collected from 12-week-old BXSB (n=5) and BXSB.Aicda^−/− (n=6) males. Bone marrow cells were flushed using syringes and resuspended at 5×10⁶/mL in RPMI (Gibco) supplemented with 2g/L sodium bicarbonate (Sigma), 25mM HEPES (Sigma), 2mM L-Glutamine (Sigma), 1% Penicillin-Streptomycin (Sigma), 10%FBS and 100μM 2-ME (Sigma). Splenocytes were treated with ACK lysis buffer and resuspended at 2×10⁶/mL in complete RPMI. After blocking with complete RPMI RT for 2h, cells were seeded and cultured at 37 C (5% CO₂) overnight. After washing with PBS three times and PBST three times, plates were incubated with either goat anti-mouse kappa (polyclonal, SouthernBiotech) or anti-mouse IgM (polyclonal, Bethyl) conjugated to alkaline phosphatase for 1h. Spots were developed with NBT/BCIP (Thermo Fisher) and plates were read on an automated ELISPOT reader and analyzed by the software (AID Diagnostika). The number of anti-dsDNA secreting cells was calculated by subtracting the number in the control group (no dsDNA coating) from the number in the experimental group.