Facs symphony a5 flow cytometer

T cell clones were assessed for GFP expression by flow cytometry. Cells were stained with Fixable Viability Dye eFluor 780 and the following monoclonal antibodies: TexasRed-PE conjugated anti-human CD3 (clone 7D6, Invitrogen, cat. MHCD0317); PerCP/Cyanine5.5 conjugated anti-human CD4 (clone OKT4, BioLegend, cat. 317428); BrillianViolet conjugated anti-human CD25 (clone BC56, BioLegend, cat. 302634).
HIV Gag p24 expression was evaluated by flow cytometry in CD4⁺ T cells derived from participants 603 and 5104. Cells were fixed and permeabilized using BD Cytofix/Cytoperm^™ Fixation/Permeabilization Kit (BD Biosciences, cat. 554714) and stained for Gag p24 protein with anti-HIV-1 core antigen monoclonal antibody (clone KC57, Beckman Coulter, cat. 6604665).
Cells were analyzed in a FACS Symphony A5 flow cytometer running FACS Diva software (version 8.5, BD Biosciences) and data was analyzed using FlowJo (version 10.10.0, BD Biosciences).
Only live cells, CD25^high (activation conditions) or CD25^low (resting conditions) were considered for measuring GFP or p24 fluorescence.

Samples were analyzed by flow cytometry after staining with fluorochrome-conjugated mAbs listed in Supplementary Table 1, as previously described^{39 (link)}. For ex vivo immunophenotyping experiments, frozen samples were thawed in RPMI-1640 medium supplemented with 10% FBS (Sigma-Aldrich), 1% penicillin–streptomycin, and 1% ultra-glutamine (both from Lonza) (hereafter referred to as R10), supplemented with 20 µg/mL DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the combination of mAbs listed in Supplementary Table 1, together with Zombie Aqua Fixable Viability kit (Biolegend). All mAbs were previously titrated on human PBMCs and used at the concentration giving the best signal-to-noise ratio, as described^{40 (link)}. Both surface markers and chemokine receptors were stained for 20 min at room temperature. All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488 nm; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo (FlowJo LLC) by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies)^{40 (link)}.