Microplate scintillation and luminescence counter

Rat spleens were harvested in sterile conditions. After erythrocyte lysis, splenocytes were isolated and re-suspended in complete RPMI-1640 media. In one-way MLR, LEW splenocytes (1x10⁵ cells/well) were co-cultured with either 2 μg/ml Concanavalin A (Con A, Sigma-Aldrich, St. Louis, MO USA) or allogeneic BN stimulator splenocytes that had been irradiated (2000 cGy; Gammacell^® 1000 Elite Nordion International, Ottawa, ON, Canada). Tregs were added in 1:1, 1:0.5, 1:0.2, 1:0.1 ratios to responder cells according to experimental design as described. Cells were cultured in quadruplicate in 96-well U-shaped plates for 4 days then pulsed with 1 μCi/well ³H-thymidine (³H-TdR, Perkin Elmer, Waltham, MA, USA) for 16 h and harvested over glass fiber filters. Thymidine uptake was quantified on a microplate scintillation and luminescence counter (Packard NXT, Meriden, CT, USA). Thymidine incorporation into spontaneously proliferating responders (in media alone) was the control and set as 1. Ratios of thymidine incorporation under all other conditions with respect to the control were acquired, providing stimulation indices (SI).