Anti cd3 bv786

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.

PBMCs were cultured in RPMI 1640 medium (Corning) with 10% fetal bovine serum and stimulated with anti‐CD3/CD28 (2 and 5 μg/ml, respectively, eBioscience), plus Golgiplug (BD Biosciences) for 5 h. The cells were then stained with anti‐CD3‐BV786, anti‐CD4‐BV711, anti‐CD8‐APC‐H7 (BD Biosciences), and fixed/permeabilized using BD cytofix/cytoperm kit (BD Biosciences) followed by intracellularly staining with anti‐interferon (IFN)‐γ‐allophycocyanin (APC), anti‐tumour necrosis factor (TNF)‐fluorescein isothiocyanate (FITC), anti‐interleukin (IL)‐2 phycoerythrin‐cyanin 7 (PE‐Cy7), anti‐IL‐10‐PE‐CF594 and anti‐transforming growth factor (TGF)‐β‐PE (BD Biosciences). Data acquisition and analysis were as described above.