Live dead fixable yellow or violet dye

0.5 to 2 × 10⁶ cells were washed with staining buffer (phosphate buffered saline (PBS, plus 5% FBS) and surface antigens were stained with fluorochrome-conjugated antibodies for 45 minutes at 4 °C. Antibodies were used at a 1:200 dilution in a final volume of 200 μl. Fluorochrome-conjugated murine (m) and human (h) specific antibodies used were obtained from Biolegend (mCD45, 30-F11; hCD4, OKT4; hCD8α, HIT8a; hCD45, HI30; hIL-17A, BL168; hIFNγ, 4S.B3; CD56, HCD56; hCD45RO, UCHL1; hCCR5, HEK/1/85a; hCXCR4, 12G5; hCD3; OKT3), Life Technologies (hCD3 PE-Texas Red, MCHD0317), eBiosciences (CD317/tetherin/BST2, 26F8; Ki-67, 20Raj1). All HIV infected samples were fixed with 4% paraformaldehyde in PBS for virus inactivation. Where indicated, intracellular cytokines were stained using the Foxp3 staining kit protocol (eBiosciences) after cell suspensions were stimulated for 5 hours with 50 ng ml⁻¹ phorbol-12-myristate-13-acetate (PMA, Calbiochem) and 1 μM ionomycin (Calbiochem) in the presence of 1x GolgiStop (BD Biosciences) at 37 °C. Dead cells were excluded by staining with Live/Dead fixable Yellow or Violet dye (Life Technologies). Samples were acquired on an LSRII or LSRFortessa (BD Biosciences) instrument. Flow cytometer data were analyzed using FlowJo (TreeStar).