Apc anti human cd73

Initially, CD4⁺ T cells were isolated from peripheral blood as described above and subjected to FACS of CD73⁺ and CD73⁻cells. To that aim, cells were stained with APC anti-human CD73 (BioLegend) diluted in PBS (1:20) in 100 μL final volume for 20 min at RT. Cells were then washed twice with PBS and resuspended in 500 μL-1000 μL PBS to achieve high cell concentrations (20 – 40×10⁶ cells/ml) for FACS. Cells were sorted into 15 mL conical tubes containing 1.5 mL RPMI^+/+. Cells were cultured for 24h, then infected and rested in the presence of ART to establish in vitro latency.^{48 (link)} Briefly, 100 ng of purified NL4-3-Luciferase was added per 1×10⁵ sorted cells, which were then infected by spinoculation as described above. 24h post virus exposure, 5 μM saquinavir was added to the cell cultures to suppress spreading infection. 5 days later, cells were stimulated with αCD3/αCD28 beads (or left untreated) in the presence of 30 μM raltegravir to prevent new infections. 24h after stimulation, luciferase activity was quantified using the bright glo luciferase assay system (Promega).