Cytokine Detection by ICS and ELISA
At day 5 or 7, cells were restimulated with immobilized anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) (4 hr with BrefeldinA [10 μg/ml] in the last 2 hr) or with PdBU and Ionomycin (5 hr with BrefeldinA [1 μg/ml]). After ICS FACS, data were collected on a FACSCalibur (Becton Dickinson) and analyzed with FlowJo (Tree Star). For ELISA, cells were similarly restimulated for 48 hr and supernatant was collected and analyzed for IL-4, IL-10, and IFN-γ as described before (Shoemaker et al., 2006).