Lsr fortessa 5 l flow cytometer

Lymphocytes 5 × 10⁶/mL were stimulated with 1 μg/mL PepMix in a CTL medium for 2 h and then for an additional 12 h in the presence of 1:1000 diluted Golgi plug (BD Biosciences, Franklin Lakes, NJ, USA). After incubation, cells were harvested with PBS and stained with the LIVE/DEAD Blue Dead Cell Staining Kit (Thermofisher) and antibodies to CD45RA-BB515, CD27-BV650, CD45RO-BV786, PD1-PECF594, CCR7-BV605 (BD Horizon, BD Biosciences), CD56-BV510, TIGIT (VSTM3)-PE/Cy7 (Biolegend, San Diego, CA, USA), CD8-AlexaFluor700 (Exbio, Prague, Czech Republic) and CD57—APC (BD Pharmingen). The cells were then washed with PBS, fixed using IC fixation and permeabilisation Buffer (eBioscience, San Diego, CA, USA) for 20 min and stained intracellularly with antibodies against IFN-gamma-PE, CD3-APCCy7 (Biolegend, San Diego, CA, USA) and CD4-PacificBlue (Exbio) in a permeabilisation buffer. The cells were washed and resuspended in a FACS buffer (FB-PBS containing 0.09% sodium azide, 1% BSA). The cells were measured using the BD LSR Fortessa 5 L flow cytometer (BD Biosciences). The obtained data were analysed by the FlowJo 10.5 software (TreeStar, Ashland, OR, USA). The gating strategy is shown in Figure 1.