Cd69 pe

To evaluate the expression of T cell markers, we collected cells from culture and washed them in phosphate-buffered saline (PBS). Cells were then colored with the following antibodies diluted in PBS, 1% BSA, and 0.01% Na azide (staining buffer): anti-human CD4-phycoerythrin (PE) (ImmunoTools; clone MEM-241; 1:20 dilution); CCR5-Alexa647 (BioLegend; catalog no. 313712; 1:100 dilution); CXCR4-allophycocyanin (APC) (BD Biosciences; catalog no. 555976; 1:50 dilution); HLA-DR-PE (Miltenyi; catalog no. 130-096-177; 1:100 dilution); CD69-PE (Immunotools; 1:20 dilution); CD38-PE (BD Biosciences; catalog no. 555460; 1:50 dilution); CD25-PE or -APC (ImmunoTools; clone MEM-181 or clone HI25a; 1:20 dilution); and CD54-ICAM-1-PE or APC (ImmunoTools; clone 1H4; 1:20 dilution). After staining for 30 min at 4°C, cells were washed and fixed in 4% paraformaldehyde (PFA) before being analyzed by flow cytometry. To stain HIV-1 Gag proteins in HIV-infected T cells, cells were first fixed for 10 min in 4% PFA and then washed in PBS. Beckman Coulter's anti-HIV-1 Gag antibody (clone KC-57-rhodamine or fluorescein isothiocyanate; 1:500 dilution) was diluted in staining buffer with 0.05% saponin (Sigma) and then used to stain cells for 30 min. Cells were then washed and analyzed by flow cytometry. To acquire samples, we used a BD FACSCanto II, and the results were analyzed by FlowJo 10.6 software.

To evaluate the expression of T cell markers, we collected cells from culture and washed them in phosphate-buffered saline (PBS). Cells were then colored with the following antibodies diluted in PBS, 1% BSA, and 0.01% Na azide (staining buffer): anti-human CD4-phycoerythrin (PE) (ImmunoTools; clone MEM-241; 1:20 dilution); CCR5-Alexa647 (BioLegend; catalog no. 313712; 1:100 dilution); CXCR4-allophycocyanin (APC) (BD Biosciences; catalog no. 555976; 1:50 dilution); HLA-DR-PE (Miltenyi; catalog no. 130-096-177; 1:100 dilution); CD69-PE (Immunotools; 1:20 dilution); CD38-PE (BD Biosciences; catalog no. 555460; 1:50 dilution); CD25-PE or -APC (ImmunoTools; clone MEM-181 or clone HI25a; 1:20 dilution); and CD54-ICAM-1-PE or APC (ImmunoTools; clone 1H4; 1:20 dilution). After staining for 30 min at 4°C, cells were washed and fixed in 4% paraformaldehyde (PFA) before being analyzed by flow cytometry. To stain HIV-1 Gag proteins in HIV-infected T cells, cells were first fixed for 10 min in 4% PFA and then washed in PBS. Beckman Coulter's anti-HIV-1 Gag antibody (clone KC-57-rhodamine or fluorescein isothiocyanate; 1:500 dilution) was diluted in staining buffer with 0.05% saponin (Sigma) and then used to stain cells for 30 min. Cells were then washed and analyzed by flow cytometry. To acquire samples, we used a BD FACSCanto II, and the results were analyzed by FlowJo 10.6 software.