Anti p24 fitc

VSV-G pseudotyped viruses (pNL4.3-deltaEnv-nLuc-2ANef-VSVG) were produced by cotransfecting pNL4.3-deltaEnv-nLuc-2ANef and pCMV-VSVG into 293T cells. Cell supernatants were collected and filtered with a 0.22 μm filter after 2 days. Viruses were aliquoted and stored at −80 °C. T_CM were generated as explained in the above section. At days 3 or 4 postisolation, cells were infected with pseudotyped viruses (pNL4.3-deltaEnv-nLuc-2ANef-VSVG) using spinoculation method. After 2 days, HIV-1-infected cells were analyzed by flow cytometry by staining with Fixable Viability Dye eFluor 450 (eBioscience, Cat. No. 65-0863-14) and anti-CD4-APC (eBioscience, Cat. No. 17-0149-41) for 30 min. After washing with 1X PBS containing 3% FBS, cells were fixed and permeabilized using Cytofix/Cytoperm (BD, East Rutherford, NJ, USA, Cat. No. 554714) for 30 min and then stained with anti-p24-FITC (KC57, Beckman Coulter, Pasadena, CA, USA, Cat. No. 6604665). At day 17, CD4⁺ T cells were isolated using Dynabeads CD4⁺ T-cell positive isolation Kit. At day 18, cells were seeded into 96-well plates, pretreated for 2 h with NAC (10 mM) or vehicle control (tissue culture media) and then treated with EPH334 (2.5 μM) or vehicle control (DMSO) for 24 h. Luciferase of supernatant (Nano-Luc) was measured using Nano-Glo Luciferase assay system (Promega, Madison, WI, USA, Cat. No. N1110).

To compare infection kinetics and depletion in collagen and suspension cultures, cultures were first treated with collagenase I (100 U, Worthington) for 30 min at 37 °C to yield cell suspensions. Cells were washed in PBS and stained with fixable viability dye 450 (eBioscience, 1:1000 in PBS) for 30 min at 4 °C, were washed in MACS buffer (PBS, 2 mM EDTA, 0.5% inactivated FCS) and subsequently stained with anti-CD8-PE Vio770 (1:100, Miltenyi Biotec, 130-096-556) and anti-CD3-PE (1:100, biolegend, 317308) for 30 min at 4 °C. After washing, cells were fixed in 3% PFA/PBS for 90 min. To detect intracellular p24, cells were permeabilized and stained with anti-p24-FITC (1:100 KC57 Beckmann Coulter, 6604665) in 0.1% Triton-X-100/PBS for 30 min at 4 °C. Cells were washed in MACS buffer and for absolute cell quantification cell counting beads (biolegend) were added prior to analysis with a FACSVerse (BD) and FlowJo software (Tree Star). CD4 T cells were identified as CD3 positive/CD8 negative cells. Relative depletion was calculated by correlating the frequency of CD4 T cells in the respective sample to the frequency of CD4 T cells in T20 controls, which was set to 100%.

The following human monoclonal antibodies (mAbs) anti CD11b PE (clone ICRF44), CD11b FITC (clone ICRF44), CD14 PerCP (clone MoP9), CD4 PE (clone RPA-T4), CD3 PerCP (clone SK7), CD80 PE (L307.4), CD86 APC (2331 (FUN-1)), CD16 FITC (clone 3G8), CD32 APC (clone 8.26) were obtained from BD Pharmingen. Anti-CD169 APC (clone 7–239), CD195 FITC (clone HEK/1/85a), HLA-A,B,C PE (clone W6/32), HLA-DR, DP, DQ FITC (clone Tu39), FITC Annexin V Apoptosis kit with 7AAD were obtained from BioLegend. Quantum Simply Cellular anti-mouse IgG microbeads were obtained from Bangs Laboratories. Anti-p24-FITC and anti-p24-RD1 were purchased from Beckman Coulter. Fixation and permeabilization buffers (Reagents A and B) were from Caltag.