Sodium l glutamine

PBMCs were thawed, washed using RPMI 1640 (Thermo Fisher Scientific), and rested for 1 hour in 0.25 μL/mL DNase I (Roche Diagnostics) containing R-10 complete medium (RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin G, 100 L/mL streptomycin sulfate [Thermo Fisher Scientific], and 1.7 mM sodium L-glutamine [Lonza]).

PBMCs, 1 × 10⁶ cells/mL, were stained at 37°C for 20 minutes with 0.5 μM CellTrace carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were washed twice with RPMI-1640 medium (RPMI) supplemented with 10% FBS (Sigma-Aldrich), and plated in 96-well round-bottom polystyrene plates, 200 μL per well. Experimental samples were incubated with 20 ng/mL of an overlapped HIV (Gag)-specific peptide pool (NIH AIDS Reagent Program). Positive control well was stimulated with 2 μg/mL of SEB (Sigma-Aldrich) and the negative control contained unstimulated PBMCs. Subsequently, cells were cultured for 5 days in R-10 medium (RPMI supplemented with 10% FBS, 100 U/mL penicillin G, 100 μL/mL streptomycin sulfate [Thermo Fisher Scientific], 1.7 mM sodium L-glutamine [Lonza] and 50 IU/mL IL-2 [R&D Systems]). On day 5, cells were collected and stained for viability using Violet LIVE/DEAD Cell Stain kit (Invitrogen) and anti-CD3-APC-H7 (clone SK7; BD Biosciences) and anti-CD8-PE (clone RPA-T8; Biolegend). Finally, cells were washed and fixed for 20 minutes at 4°C with 4% paraformaldehyde solution (PFA; Sigma-Aldrich). Multiparametric flow cytometry analyses were performed on an LRS Fortessa flow cytometer using FACS Diva software (BD Biosciences). Data were analyzed using the FlowJo 10.7.1 software (Treestar).