Cd8 fitc clone sk1

The expression of ectonucleotidases (CD39 and CD73) on αβDNT and activated Treg cells was evaluated by flow cytometry. The gating strategy for activated Treg cells was performed as previously described (5 (link)). PBMCs were stained with directly conjugated antibodies for 30 min at 4°C in the dark. The cells were washed before flow cytometry analysis. Antibodies used included anti-human CD3-BV785 (clone SK7), CD4-APC-fire750 (clone SK3), αβTCR-BV421 (clone IP26), CD56-BV510 (clone HCD56), CCR7 (CD197)-PE-cy7 (clone G043H7), HLA-DR-AF700 (clone L243), CD73-PE (clone AD2), CD39-BV605 (clone A1, BioLegend, San Diego, CA, USA), CD45RA-BV711 (clone HI100), CD38-BUV737 (clone HB7), CD25-PE-CF594 (clone M-A251), CD8-FITC (clone SK1), CD8-BUV395 (clone RPA-T8, BD Biosciences, San Diego, CA, USA), and the corresponding isotype controls. Data were acquired on a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

To assess CD8⁺ T-cell degranulation, purified CD8⁺ T cells were incubated in the presence or absence of P815 target cells at a 1:1 ratio for 30, 60, 90, 120, and 180 min at 37 °C. Cells were then stained using the following fluorescently labeled antibodies: anti-CD107a-PE (clone H4A3), CD56-APC (clone NCAM16.2), CD8-FITC (clone SK1), and CD3-PerCP (clone SK7), all of which were from BD Biosciences (San Jose, CA). Data were collected using a Cyto-Flex-S cytometer (Beckman). CD3⁺CD8⁺ cells were gated and assessed for surface expression of CD107a.