Lsr 2 cytometer

Cells were stimulated overnight with pools of 15–amino acid peptides overlapping by 11, spanning the whole RHV polypeptide sequence (1 µg/mL). Dimethyl sulfoxide (DMSO) was applied to negative controls and phorbol 12‐myristate 13‐acetate/ionomycin to positive controls. Mouse antirat cluster of differentiation 28 (CD28; BD Pharmingen, 1:62.5) was added to each well. One million to 3 million PBMCs per well were incubated with peptide pools or controls (2 hours, 37°C) then Golgi Plug (BD Pharmingen; 1:1,000, 16‐18 hours, 37°C). After incubation with mouse antirat CD32 (BD Pharmingen; 1:100, RT, 15 minutes), a surface‐staining cocktail was applied (30 minutes) (Supporting Table S3), followed by fixation and permeabilization (Cytofix/Cytoperm; BD Biosciences), cell washing (Perm/Wash buffer; BD Biosciences), and intracellular staining (30 minutes) (Supporting Table S1). Cells were acquired on an LSR II cytometer, and the results were analyzed using FlowJo software (version 10).

αSMA was quantified by flow cytometry in cultures, with or without 5 ng/mL of TGFβ, and/or 100 nM of substance P. Cells were trypsinized, fixed in 70% ethanol, and treated with acid, as described for BrdU staining above. 50 µL of anti-αSMA-Alexa 488 (abcam, Burlingame, CA, USA, Catalog # ab202295) at a dilution of 1:500 was added to the cell pellets after neutralization. Data were collected on the LSRII cytometer and analyzed using Flowjo software version 10.10.0.

HLACs were infected with a X4 virus by spinoculation as explained before. After 3 days, uninfected cells were labeled with CellTrace CFSE (2 µM, Invitrogen) for 5 min at room temperature. Uninfected-CFSE⁺ (0.6 × 10⁶ cells) were pre-treated for 2 h with or without autophagy inhibitors at 37 °C. Then, infected or uninfected CFSE^- cells were added to the treated CFSE⁺ target cells in a ratio 1:1, incubated in the presence or absence of drugs for 2 days at 37 °C, collected and analyzed by flow cytometry. Cells were stained with the antibodies CD3-APC-Cy7, CD4-APC and CD8-PerCP (BioLegend), fixed and permeabilized (FIX & PERM kit, Thermo Fisher) and stained with anti-p24 antibody (KC57-PE, Coulter). To calculate CD4⁺ T cell depletion, total live lymphocytes were first gated according to forward and side scatter and then gated on the CD3⁺ population. Next, CFSE⁺ cells were selected, CD4⁺ and CD8⁺ T cells were gated and CD4⁺/CD8⁺ ratio was obtained. Cell depletion was calculated as a percentage compared to the uninfected control as previously described^{22 (link)}. To analyze intracellular p24 level, CD3⁺ CFSE⁺ cells were gated and CD8-negative cells were selected to include both CD4-positive and CD4-negative cells. Data was acquired in a LSRII cytometer and analyzed with FlowJo software.