Quantifying Intestinal IgA-Secreting Cells

The frequencies of intestinal IgA⁺ ASCs and VP6⁺ IgA⁺ ASCs were determined by ELISPOT as described (20 (link)). Briefly, 96-well plates (Millipore) were coated with affinity-purified goat anti-human IgA + IgG + IgM (H + L) (KPL) at a concentration of 4 μg/ml. Wells were coated with phosphate-buffered saline (PBS) as a negative control. Plates were incubated overnight at 4°C and blocked for 2 hours at 37°C with complete medium before use. B cells were suspended in complete medium containing peroxidase-conjugated goat anti-human IgA (Sigma-Aldrich), distributed in ELISPOT plates, and incubated for 4 hours at 37°C in 5% CO₂. Plates were washed and developed with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories). The total ASCs per well was determined by counting the spots using the ImmunoSpot Analyzer (Cellular Technology Limited). Background ASCs detected in the wells coated with PBS were subtracted from the quantities of ASCs in treated wells.