Cd45 pacific blue

ASC cultures from early passages (1–6 population doublings) were harvested and resuspended in DMEM/F12 with 2% FBS. The cells were then centrifuged and suspended in cold PBS at a concentration of 10⁶ cells/100 μl. Cell aliquots were stained with monoclonal mouse anti-human antibodies against the following antigens: CD44-peridinin-chlorophyll-protein/cyanin 5.5, CD90-phycoerthyrin (PE), PE/Cy7-CD105, CD73-fluorescein isothiocyanate, CD34-Alexa Fluor 647, CD45-Pacific Blue (PB), CD11b-PB, and CD31-PB (BioLegend, San Diego, CA) for 30 minutes in the absence of light at 4°C. The cells that were stained with a single antibody coupled with a fluorescent dye were acquired for compensation purposes. After incubation, the cells were washed and resuspended in cold PBS. Flow cytometry was performed using a Becton Dickinson LSR II. A gate was set to include only the viable propidium iodide-negative cells. The number of cells staining positive for a given cell surface marker was determined by the percentage of cells present within an established gate. A minimum of 10,000 events were counted for each analysis.

Freshly harvested stromal vascular cell (SVC) human lipoaspirate cell pellets were resuspended in FACS buffer and stained with antibodies specific to human: CD45‐Pacific blue (PB, BioLegend, San Diego, California, Cat#304022) for hematopoietic cells; CD235a(Glycophorin A)‐eFluor 450 (BioLegend, Cat#349108) for erythrocytes, and erythroid progenitors; CD31(PECAM‐1 or platelet endothelial cell adhesion molecule)‐eFluor 450 (BioLegend, Cat#303114) for endothelial cells; CD34‐AlexaFluor(AF)‐488 (BioLegend, Cat#343517) for ASCs; and CD146‐PE/Cy7 (BioLegend, Cat#361007) for the proangiogenic ASC subset. All primary antibodies were stained in a 1:100 ratio, which was determined based on prior experiments. 4′,6‐diamidino‐2‐phenylindole (DAPI, Thermo Fisher Scientific, Cat#D1306) was used as a live‐dead stain. Flow cytometry (FACS Aria II, BD Biosciences, San Jose, California) was used to isolate three populations of ASCs based on a Lin‐negative (CD45−CD235a−CD31−) and positive gating strategy; CD34+CD146+, CD34+CD146−, and CD34+ unfractionated (UF) ASCs (Figures 1 and S1).¹⁵, ¹⁶, ¹⁷ Cells were sorted using a 100 μm nozzle, and a flow rate of 1, with the “purity” setting. Purity was tested before sorting by analyzing 2000 freshly sorted cells and was found to be >99% for each of the three ASC subpopulations.

Human ASCs were isolated from the SVF according to surface marker expression profiles by FACS. The SVF pellet was resuspended in 100 μL for staining with the following anti‐human fluorescent antibodies: CD45‐Pacific Blue (PB) (#304029, Biolegend, San Diego, California) to label hematopoietic cells; CD235a(Glycophorin A)‐eFluor450 (#48‐9987‐42, eBioscience, San Diego, California) to label erythrocytes and erythroid progenitors; CD31(PECAM‐1)‐eFluor450 (#48‐0319‐42, eBioscience) to label endothelial cells; CD34‐Fluorescein Isothiocyanate(FitC) (#555821, BD Biosceinces, San Jose, California) to label ASCs; and CD74‐Allophycocyanin(APC) (#326811, BioLegend) to label the antifibrotic subset of ASCs. All primary antibodies were diluted 1:100. After 30 minutes incubation with primary antibodies shielded from light, cell suspension solutions were diluted with FACS buffer, centrifuged (450g, 5 minutes, 4°C), resuspended in 500 μL of FACS buffer, and filtered through a 70 μm nylon mesh. 4',6‐diamidino‐2‐phenylindole (DAPI) (#62248, ThermoFisher) was added as a live‐dead stain (1:10 000). FACS (BD II Aria) was then used to isolate ASCs (CD45−, CD235a−, CD31−, CD34+ live single cells) that were CD74+ (CD45−, CD235a−, CD31−, CD34+, CD74+), CD74− (CD45−, CD235a−, CD31−, CD34+, CD74−), and “unsorted” (US, CD45−, CD235a−, CD31−, CD34+ only).