αcd3 apc h7

Enriched cells were resuspended in 50 µl Cell Staining Buffer (CSB) (420201, Biolegend, USA) plus 10% FcR-Block (130-059-901, Miltenyi) then incubated for 15 min at 4°C. Cells were labelled with biotinylated antibodies against TCR-γᵹ (130-113-502, Miltenyi), TCR-Vα24 (130-117-958, Miltenyi) and TCR Vα7.2 (130-110-957, Miltenyi) in a total volume of 100 µl of CSB for 15 min at 4°C. After washing in 2 ml CSB, cells were stained for 30 min at 4°C with α-biotin FITC (130-110-957, Miltenyi), αCD161 PE (339904, BioLegend), αCD25 PC5.5 (356112, BioLegend), αCD56 APC (318310, BioLegend), αCD3 APC-H7 (560176, BD Biosciences, USA) and αCD4 BV421 (344632, BioLegend) in a final volume of 100 µl/sample comprising CSB plus 50 µl Brilliant Staining Buffer (563794, BD). Cells were washed with 2 ml CSB then resuspended in 500 µl PBS prior to sorting on a FACSAria Fusion (BD).

The following mAb conjugates were used at pre-titrated concentrations: (i) αCD3-APC-H7 (BD Biosciences, San Jose, CA, USA); (ii) αCD8-QD705, αCD14-Pacific Blue and αCD19-Pacific Blue (Life Technologies); (iii) αCD8-PE, αCD57-FITC and αCCR7-PE-Cy7 (BD Pharmingen, San Jose, CA, USA); and (iv) αCD27-PE-Cy5, αCD45RA-ECD and αCD45RO-ECD (Beckman Coulter, High Wycombe, UK). LIVE/DEAD Fixable Aqua and Violet Dead Cell Stain Kits (Life Technologies) were used to eliminate nonviable cells from the analysis. Peptide-major histocompatibility complex dextramers conjugated separately to APC and PE (Immudex, Copenhagen, Denmark) were used for magnetic enrichment.