Flow cytometry IFN-γ cytokine secretion assay.

A mixture of draining lymph node (axillary/brachial) cells from CCR4^–/– and CCR4^+/+ airway allograft recipients on day 7 was collected for ex vivo stimulation. Single-cell suspension was prepared as described above, and 1 × 10⁶ recipient cells from CCR4^–/–and CCR4^+/+ allograft recipients and naive nontransplanted CCR4^–/–and CCR4^+/+ mice were stimulated in the presence of either RPMI 1640 supplemented with 5% FBS alone or 2 × 10⁶ irradiated and digested BALB/c splenocytes. After 16 hours of incubation at 37°C and 5% CO₂, cells were washed and analyzed using the flow cytometry IFN-γ cytokine secretion (FCIS) assay (Miltenyi Biotec) via flow cytometry analysis on a minimum of 250,000 total events. The cells were stained for 7-AAD (to exclude dead cells in the PerCP channel), B220-PerCP (to exclude B cells), CD3-FITC, IFN-γ–PE, and either CD4-APC or CD8-APC (to measure percent cell frequency of alloresponsive T cells). The coefficient of variation with FCIS is 5%–15%, with a lower limit of detection of 0.01% or 1/10,000 IFN-γ–secreting cells (63).