Cell labeling and flow cytometry

OVA-H2K^b-BW5147 cells, H2K^b-BW5147 cells, OT-I splenocytes, and C57BL/6 splenocytes were stained for 30 min at 37°C using 20 μM of CMTMR (CellTracker^™ Orange CMTMR Dye, Thermo Fisher Scientific), 50 μM of CMAC (CellTracker^™ Blue CMAC Dye, Thermo Fisher Scientific), 1 μM of CFSE (CellTrace^™ CFSE Cell Proliferation Kit, Thermo Fisher Scientific), and 2 μM of Far Red (CellTrace^™ Far Red Cell Proliferation Kit, Thermo Fisher Scientific), respectively. Flow cytometry analysis was performed with NovoCyte^® (ACEA Biosciences), followed by sorting of the cells using FACS with an FACSAria^™ II Cell Sorter (BD Biosciences) or On-chip Sort [32] (On-chip Biotechnologies) system. Cell sorting was performed using the following settings: FACSAria^™ II, pressure = 70 psi; sorting speed = ca. 1000 events per second; nozzle diameter = 70 μm. On-chip Sort, pressure = 0.8 kPa (ca. 0.3 psi); sorting speed = ca. 100 events per second; fluidics channel diameter = 80 μm. Data analysis was performed using NovoExpress (Agilent) and FlowJo softwares (BD Biosciences).