ELISPOT Assay

Anti-GPI antibody secreting cells were measured by ELISpot as described (36). Briefly, cells from the joint draining lymph nodes (axillary, brachial, and popliteal LNs) from 6 week-old KRN.g7, IDO1 ko KRN.g7, IDO2 ko KRN.g7, and dko KRN.g7 mice were plated at 4 × 10⁵ cells per well and diluted serially 1:4 in Multiscreen HA mixed cellulose ester membrane plates (Millipore) coated with GPI-his (10 μg/ml). The cells were incubated on the Ag-coated plates for 4 h at 37°C. The Ig secreted by the plated cells was detected by Alkaline Phosphatase-conjugated goat anti-mouse total Ig secondary Ab (Southern Biotechnology Associates) and visualized using NBT/BCIP substrate (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; Sigma).