Cd45 bb515

Single cell suspensions extracted from the lungs were stained with a live/dead-Infrared marker (Thermo Fisher) and incubated with monoclonal antibody CD16/CD32 Becton Dickinson Biosciences (BD) to block Fc receptors, before adding a cocktail of directly conjugated mAbs directed against the following surface markers; CD11b-BV711 (M1/70, 563168), Ly6G-PerCp-Cy5.5 (1A8, 560602), CD45-BB515 (30-F11, 564590), SiglecF-PE-CF594 (E50-2440, 562757), CD103-BV421 (M290, 562771), CD11c-BV510 (HL3, 562949), CD3e-BV786 (145-2C11, 564379), CD19-BV605 (1D3, 563148), CD335-BV605 (29A1.4, 560469), and Ly6C-APC (AL-21, 560595) all purchased from BD and FceR1-PE-Cy7 (MAR-1, 25-5898-82), MHC-II-AF700 (M5/114.15.2, 56-5321-82) from Thermo Fisher (ebioscience). Data acquisition was performed with the BD LSR Fortessa (BD) and Diva software, and the analysis was performed with FlowJO X software (TreeStar, Ashland, OR). In initial control experiments, the right lobes were evaluated for the frequency of contaminating peripheral blood mononuclear cells after cardiac PBS infusion by staining with an anti-CD115 mAb (T38-320, 565249, BD), which is uniformly expressed on all circulating blood monocytes (13 (link)) but not on lung monocytes (14 (link)). The frequency of CD115⁺ cells was below 0.01% among the lung cells, arguing that we had little if any blood leukocyte contamination (Figure S1B).

In order to identify human EPCs, 2 × 10⁵ cells were suspended in 50 μL fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.5% FBS) then stained with 0.2 mg/mL of each of the following anti-human antibodies: CD31-PE (LifeSpanBioSciences, Seattle, WA, USA), CD34-BV421 (BD Biosciences, San Jose, CA, USA), CD45-BB515 (BD Biosciences), VEGFR-2 PE (R&D Systems, Minneapolis, MN, USA), CD14-FITC (BD Biosciences), and CXCR4-PE (BioLegend, San Diego, CA, USA). OneComp eBeads (Carlsbad, CA, USA) were first stained with 1 μL of each different fluorochrome then used as single-color compensation controls. An unstained sample served as a reference for positive staining. Cells were analyzed using a Cyan flow cytometry (Beckman Coulter, Brea, CA, USA). Single cell data was analyzed using FlowJo, LLC software that gave a percentage for positive cells.

The FACSLyric was used as a flow cytometer for counting CD4 in people living without HIV and in people living with HIV. A tube containing a mixture of 200 µL of peripheral blood, 5 µL of BB515-CD45 (BD Biosciences, Franklin Lakes, NJ, USA), and 5 µL of PE-CD4 (BD Biosciences, NJ, USA) was incubated at room temperature in the dark for 15 min. After staining the cells, the mixture was added to 1 mL of Lysing solution OptiLyse C (BD Biosciences, NJ, USA) and then incubated for 10 min. The cells were then centrifuged at 2000× g rpm for 5 min after adding 5 mL of PBS (Phosphate-buffered saline, pH 7.4). The supernatant was removed and the cells resuspended in 1 mL PBS. The resuspended sample (1 mL) was then loaded into the flow cytometer and the CD4/CD45 ratio calculated using flow cytometry. The CD4 gating strategy was performed with reference to a previous study [22 (link)].