Pe anti human cd69

Flow cytometry experiments were performed according to the procedure described earlier [15 (link)]. In particular, cells suspensions were incubated with APC-Cy7 live/dead (BD, 1:100), PC5.5 anti-human CD4 (BD, 1:100), BV421 anti-human TNF-α (BD, 1:100), BV605 anti-human CD8 (BD, 1:100), BV650 anti-human IFNγ (BD, 1:100), APC anti-human IL2 (BD, 1:100), AF700 anti-human GZMB (BD, 1:100), PE anti-human CD69 (BD, 1:100), and BV510 anti-human CD3 (BD, 1:100) antibodies. Flow cytometry was performed using a BD Fortessa FACS with Diva software v6.0. and results were quantified using FlowJo V10 software.

For flow cytometry assay, samples were suspended and adjusted to standard concentrations according to specifications. All fluochrome-conjugated Abs, unless specifically stated, were purchased from eBiosicience. The PE anti-human CD69, APC-anti-human FoxP3, and human Foxp3 staining buffer sets were purchased from BD (USA). For magnetic cell separation, PBMCs were separated from whole blood using Lymphoreo (Fresenius Kabi Norge AS, Norway). Then the proper volume (10 μl per 10⁶ cells) of anti-CD4 or anti-CD8 microbeads (Miltenyi, De) was added to the cell suspension and allowed to react on ice for 15 min before the cells went through a magnetic separator. The magnetic separator was washed three times to thoroughly separate CD4⁻ or CD8⁻ cells from CD4⁺ or CD8⁺ cells. The positive cells were then collected from the magnetic separator.