Anti cd8 fitc clone rpat8

Following stimulation, cells were collected and washed with PBS. Firstly, cells were labeled with the Zombie NIR Fixable Viability Kit (BioLegend) at room temperature for 20 min to distinguish live cells. Then, cells were washed with FACS washing buffer (PBS supplemented with 0.2% BSA and 0.09% sodium azide) and stained with anti-CD3-BV605 (clone SP34-2, BD Biosciences) and anti-CD8-FITC (clone RPAT8, BD Biosciences) at 4 °C for 30 min, then fixed/permeabilized with Fixation and Permeabilization Solution (BD Biosciences) at 4 °C for 20 min. After fixation, cells were washed by Perm/Wash Buffer (BD Biosciences) and stained with anti-IFN-γ-BV711 (clone 4S.B3, BioLegend), anti-MIP-1β-BV421 (clone D21-1351, BD Biosciences), anti-IL-2-PE-Cy7 (clone MQ1-17H12, BioLegend), or anti-TNF-α-BV650 (clone MAb11, BioLegend) at 4 °C for 30 min. Finally, cells were washed by Perm/Wash Buffer and fixed in 1% paraformaldehyde and analyzed on BD FACSAria II within 24 h.

Peripheral blood mononuclear cells (PBMC) were freshly isolated by Ficoll (GE Healthcare Biosciences) as described previously [37 (link)]. PBMC subpopulations were sorted with anti-CD4, anti-CD8, anti-CD14, anti-CD19, and anti-CD56 MicroBeads (Miltenyi Biotec) with an autoMACS Pro Separator (Miltenyi Biotec) according to manufacturer’s instructions. The purity of sorted cells was checked by flow cytometry with the following antibodies: anti-CD4 APC-H7 (clone SK3, BD Biosciences), anti-CD4 ECD (clone SFCI12T4D11, Beckman Coulter), anti-CD8 FITC (clone RPA-T8, BD Biosciences), anti-CD11c APC (clone B-ly6, BD Biosciences), anti-CD14 PB (clone M5E2, BD Biosciences), anti-CD19 PE (clone HIB19, eBioscience), and anti-CD56 PE-Cy7 (clone NCAM16.2, BD Biosciences) on a LSRII flow cytometer (BD Biosciences). We did not pursue the proposed experiments if the purity of sorted cells was less than 90 %. Analyses were performed using FlowJo software (Treestar).