Cd90 alexa fluor 647

Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD8-FITC, CD4-FITC, CD4-PE, CD3-APC, CD3-APC-Cy7 (eBioscience, San Diego, CA), CCR5-PE-Cy7 (BD Biosciences, San Jose, CA), CCR5-PE (R&D Systems, Minneapolis, MN), CCR6-PerCp-Cy5.5, CD90 Alexa Fluor-647 (Biolegend, San Diego, CA). For the HIV-infection experiments, following infection for 6–7 days, intracellular levels of p24 were analyzed as described previously^{8 (link)}. Briefly, after surface staining, cells were washed, fixed and permeabilized (20 min) following instructions provided in the Cytofix/Cytoperm Plus kit (BD Biosciences) and stained for intracellular p24 with KC57-FITC antibody (Beckman Coulter; Danvers, MA) for 30 min. Analysis was performed on BD FACSCalibur or BD FACSCanto flow cytometers (BD Biosciences) using FACSdiva software, and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers was measured by the percentage of positive cells and the mean fluorescence intensity (MFI).

Cultured fibroblasts (passages 0-2) were grown in culture to 80% to 90% confluence and trypsinized, as previously described.^{4 (link)} Single-cell suspensions were aliquoted to a 96-well plate. Cells were stained with the following antibody panel: LIVE/DEAD Fixable Aqua, CD31 (PECAM) Brilliant Violet 421 (303124; BioLegend), CD34 PerCP-CY5.5 (343612; BioLegend), FAPa PE (FAB3715P-025; R&D Systems), CD90 Alexa Fluor 647 (328120; BioLegend), and PDGFR (platelet-derived growth factor receptor) PE/Cy7 (323508; BioLegend). After staining, cells were fixed and permeabilized. Single-stained and florescence minus one–stained samples were used as controls for gate selection. Viability Aqua–negative (live) cells were subsequently evaluated by percentage population of fibroblasts (FAP⁺). Fibroblast cells line were grouped according to etiology (n = 3), with rapidly processed autopsy tracheal mucosa–derived nonscar fibroblasts used as controls. All analyses were performed in FlowJo Flow Cytometry Analysis Software (Treestar, Version 10.7.1 for Mac).