Cell trace yellow proliferation dye

To analyze productively infected cells, 2.5x10⁵ cells were stained for CD4 (clone: S3.5, APC, BD), fixable viability dye (eF450, eBiosciences) and intracellular p24-gag (BD) and were analyzed on a Celesta flow cytometer (BD) as previously performed [34 (link)]. For proliferation studies, cells were stained for CD4, viability and intracellular Ki67 (clone: 6604665, FITC, Biolegend). To analyze cellular RNA/ DNA content in order to determine cell cycle state, cells were stained with 7-AAD (Millipore Sigma, MA) and PyroninY (Sigma, MO) as previously done [84 (link)]. To label cells prior to co-culture assays, cells were stained with Cell Trace Yellow proliferation dye (ThermoFisher, MA). Flow cytometry analysis was performed in FlowJo software (BD) and further statistical analysis was performed in GraphPad Prism (CA).

To analyze productively infected cells, 2.5 × 10⁵ cells were stained for CD4 (clone S3.5, APC; BD), fixable viability dye (eF450; eBiosciences), and intracellular p24-Gag (Kc57, FITC; BD) and were analyzed on a Celesta flow cytometer (BD) as previously performed, with 1.5 × 10⁵ events collected per condition (35 (link)). To label cells prior to coculture assays, cells were stained with Cell Trace Yellow proliferation dye (ThermoFisher, MA) according to the manufacturer’s instructions. To determine percent expression of total and phosphorylated SAMHD1, CD4 T cells were stained with pSAMHD1 (Cell Signaling Technologies) and total SAMHD1 (Origene) according to manufacturer protocols. For determination of CD4 and coreceptor MFI, cells were stained for CD4 (clone S3.5, APC; BD), fixable viability dye (eF450; eBiosciences), CXCR4 (PE-Cy7; eBiosciences), and CCR5 (BV786; BioLegend) and were analyzed on a Celesta flow cytometer (BD) as previously performed (35 (link)).