Quantifying Cytokine-Producing Cells Using ELISPOT

ELISPOT plates (Millipore, Bedford, MA) were precoated with 5µg/mL of capture antibodies against gamma-IFN or IL-17 (Mabtech, Nacka Strand, Sweden) in phosphate-buffered saline (PBS) and stored overnight at 4°C. The responding cells separated from fresh PBMCs were co-cultured with an equal number of irradiated donor or autologous PBMCs as stimulating cells (1.5 × 10⁵ cells/well), or unstimulated in medium alone, or with phytohemaglutinin at 1 µg/mL (Sigma–Aldrich Corp, St. Louis, MO). After 44 h incubation at 37°C, the plates were washed and biotinylated detection antibodies (Mabtech) were added for 2 h at room temperature. After five washes with PBS, streptavidin–horseradish-peroxidase conjugate in PBS BSA 0.5% (Dako, Glostrup, Denmark) was added for 1 h at room temperature, followed by five washes. Finally, 100 µL/well of 3,3,5,5-tetramethylbenzidine liquid substrate (Sigma–Aldrich) was added and incubated for 15 min in the dark. The resulting spots were counted with an ELISPOT image analyzer (CTL, Inc., Cleveland, OH), as previously described (7 ,19 (link),20 (link)).