Aria flow cytometer

For cell sorting, 5x10⁷ OVCAR3 cells were stained with 50μg of mAb216-Alex Fluor 488 [less than saturation concentration] and PI for 1 hour at room temperature, washed three times with PBS, and sorted on a BD Aria flow cytometer at Stanford FACS facility. FCS files are available from the Stanford FACS facility

Immortalized human bronchial epithelial cells (iHBECs; a gift from Dr. Gisli Jenkins, University of Nottingham, U.K.) were transduced with mStrawberry-Luc lentivirus to create a stable iHBEC cell line co-expressing mStrawberry fluorescent protein and luciferase. Lentivirus production was as previously described [32] (link) but created with the pLV-LS1 transfer vector and the packaging and envelope vectors pCMV-dR8.74 and pMD.G2 (Plasmid Factory). Briefly, viral supernatants were created by co-transfecting 293T HEK cells with the above plasmids using JetPEI (Polyplus Transfection). Supernatant containing lentivirus was collected at 48 and 72 h and was concentrated by ultracentrifugation. Each collection underwent ultracentrifugation to concentrate virus. To generate a transduced cell line, iHBECs were grown in LHC9 medium (Gibco), seeded at 1 × 10⁵ cells in a T25 flask and transduced at an MOI of 7.5 with 4 μg/ml polybrene (Sigma). After 8 h, medium was changed and cells were passaged when approximately 80% confluent. To obtain a pure luciferase-expressing population, mStrawberry fluorescent protein-expressing cells were sorted by fluorescence-activated cell sorting (FACS) using a BD Aria flow cytometer.

Single cell suspension was collected from various tissues (hLNs, lungs, and spleens) from sacrificed mice. Surface CD markers were stained at 4°C for 20 min, protected from light. After according treatments, intracellular staining was carried out following surface staining, 4% PFA/PBS fixation and 0.2% TritonX-100 permeabilization. Data were acquired with BD Aria flow cytometer (BD Biosciences, USA) and analyzed by Flowjo (Treestar). Cells were sorted by BD Aria III flow cytometer (BD Biosciences, USA).

Blood-drawing from participants was performed on day 0 and within a week after TRT (Fig. 1a). Cytokines (IL-2, IFN-γ) in serum were detected by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, MN, USA) following the manufacturer’s instruction. For assay of T cell populations and NK cells, 100 μL of EDTA anticoagulant blood samples were stained with corresponding antibodies (BD Bioscience), namely, anti-CD3⁺, CD4⁺ and CD8⁺ for T cells, anti-CD3⁺ and CD56⁺ for NK cells, in darkness for 20 min. Then, erythrocyte lysis buffer was added. After being vortexed for 15 s and incubated at room temperature for 5 min, the samples were centrifuged to remove the supernatant and washed with PBS. After being re-suspended with staining buffer, the samples were analyzed on the BD Aria flow cytometer (BD Bioscience).

A whole testis dissociate was prepared using a two-step enzymatic digestion [45 (link)]. For MCAM staining, testes from Gfra1-creER^T2; tdTomato mice (n = 3) were dissociated >3 months after tamoxifen administration. The single-cell suspensions from two testes were incubated with Alexa Fluor 647 anti-MCAM antibody (ME-9F1, BioLegend) at a concentration of 6 g/ml for 45 minutes at 4°C. After exclusion of doublets and DAPI-positive cells, Alexa Fluor 647 and tdTomato double-positive cells were gated and collected using a BD Aria flow cytometer.

Comb-mut V4 cell line was generated and characterized as described^{25 (link)}. Following a similar protocol, the MPER-TM_654-709 cell line was developed. Briefly, a PCR amplicon encoding a TPA leader sequence, codon-optimized MPER-TM (654-709) of BG505, followed by a stop codon was cloned into NotI and XhoI sites of pLenti-III-HA (Applied Biological Materials). The resulting MPER-TM lentiviral vector was used to generate lentiviral particles, which were then used to transduce 293T cells following the manufacturer’s instructions. The transduced cells were cultured in medium containing 10 μg/ml puromycin to select for stable integrants and were sorted on a BD Aria flow cytometer to select the 10E8 (high) stable cell line MPER-TM_654-709.
For flow cytometry experiments, a total of 10⁷ cells of stable cell lines HIV-1 MPER-TM_654-709 or Comb-mut Env (V4) were washed in PBS and labeled with Fixable Aqua Dead Cell Stain (Life Technologies). Cells were washed in FACS buffer (PBS supplemented with 2% heat-inactivated fetal bovinse serum) and were stained with monoclonal antibody. After another wash, cells were stained using APC-conjugated mouse anti-human Fc (BioLegend HP6017). Soluble CD4 was incubated with cells for 30 min prior to staining cells with antibody. Cells were acquired and analyzed by using NovoCyte (ACEA Biosciences). Data were analyzed using FlowJo software (Tree Star).