Live dead fixable viability stain 510

For surface staining, PBMCs were isolated from HC and HIV-1-infected patients with or without syphilis infection. Cells were washed with 1% bovine serum albumin in PBS and were labeled with LIVE/DEAD fixable viability stain 510 (BD Biosciences, San Jose, CA, USA), and dead cells were excluded. Then, cells labeled with specific surface antibodies: gating strategy on CD3⁺γδTCR⁺ cells, Vδ₁ and Vδ₂ T cells, or γδT_Naive (CD27⁺CD45RA⁺), γδT_CM (CD27⁺CD45RA⁻), γδT_EM (CD27⁻CD45RA⁻), and γδT_EMRA (CD27⁻CD45RA⁺) were displayed (Figure 2). Cytometer setup and tracking calibration particles were used to ensure that fluorescence intensity measurement was consistent in all experiments. Flow cytometry Comp-Beads kits (BD Biosciences, San Jose, CA, USA) were used for compensation. Forward angle and side scatter light gating were gated on lymphocytes and were used to exclude cell debris from the analysis. Forward height and forward area were used to exclude doublet cells. Cells were performed with a FACScan flow cytometer, as previously described (33 (link), 34 (link)). The final analysis was performed with FlowJo software (version 7.6.2; Tree Star Inc., Ashland, OR, USA), which generated a graphical output. The strategies for the analysis of flow cytometry data are detailed in Figure 2.

The following antibody clones were used for staining the cells:
DCs: Lineage FITC, HLADR PerCP (clone L-243), CD11c APC (cloneBu15), CD123 BV421 (clone 6H6), CD86 (clone IT2.2), and CD14 BV650 (cloneM5E2) were obtained from BioLegend (San Diego, CA). Live/Dead Fixable Viability Stain 510 was from BD Pharmingen (San Jose, CA, USA).
CD8T: CD8 PerCP (clone-SK1), perforin FITC (cloneB-D48), CD107aPE (clone H4A3), and granzyme B AL647 (clone GB11) were obtained from BioLegend.
Viruses: Virus-irradiated and heat-inactivated SARS-CoV-2 Isolate USA-WA1/2020 and control irradiated Vero cell lysate were obtained from Bei Resources (NIAID). As per the BEI Resources, gamma irradiation was performed using (5 × 106 RADs) on dry ice, and heat inactivation was performed by heating to 65°C for 30 min. The inactivated viruses are biosafety level 1. The institutional biosafety protocol (IBC) number is 2008-1243.