Anti cd62l bv650

Based our protocol from previous publication (16 ), we resuspended the splenocytes from the immunized in 10μl of RPMI-1640 media. The cells were ex vivo stimulated with anti-CD107a antibody (BD Biosciences, 553792), anti-CD28/CD49d antibody (BD Biosciences, 347690) and OLP or DMSO in 10μl. The mixture was incubated in 5% CO₂, 37°C incubator for 1hour and treated with 4μl of mixture of the complete RPMI-1640 media: Brefeldin A (GolgiPlug, BD Biosciences, 555029):Monensin (GolgiStop, BD Biosciences, 554715) in 55:3:2. After 12hours of incubation in 5% CO₂, 37°C, the cells were washed with PBS and stained with surface antibodies (anti-CD44 BV421 [BD Biosciences, 536970], anti-CD8a BV510 [BD Biosciences, 563068], anti-CD62L BV650 [BD Biosciences, 564108], anti-CD3 BV786 [BD Biosciences, 564010], anti-CD4 PerCP-Cy5.5 [BD Biosciences, 561115], and anti-CD19 APC [BD Biosciences, 561738]) for 15 minutes at RT and washed. Cells were then fixed with 4% paraformaldehyde in PBS and permeabilized. We then washed the cells and stained with FACS antibodies (anti-IL-2 FITC [BD Biosciences, 562040], anti-TNF PE [BD Biosciences, 554419], anti-IFN-γ [BD Biosciences, 557735], and anti-CD107a [BD Biosciences, 560647]) by 20 minutes of incubation at room temperature, washed twice with the permeabilization buffer and resuspended with 300μl of PBS.