Cd235a fitc

To characterize EVs, platelet-free plasma samples were stained and acquired using 14 different fluorochrome-conjugated antibodies in three separate panels, including CD235a-FITC, CD62p-APC, CD3-PerCP/Cy5.5, CD19-A700, CD28-FITC, CD62L-APC, CD11b-PE/Cy7, CD154-APC, CD41a-PerCP/Cy5.5, CD66b-PE (Biolegend), CD15-FITC (ExAlpha), CD108a-PE, CD16-V450, and CD142-PE (BD Biosciences). Samples were washed with a 0.22-μm centrifugal filter (Millipore) at 850× g for 3 minutes. EVs were then harvested from the top of the filter after washing, and data were acquired on a flow cytometer ((BD LSR II, BD Biosciences). In addition, 27 cytokines were measured using human cytokine chemokine kits (Multiplex MAG kits, Millipore): granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, IL-10, IL-12(p70), IL-17A, IL-1β, IL-2, IL-21, IL-23, IL-6, IL-7, IL-8, CXCL11/ITAC, CCL3/MIP-1α, CCL4/MIP-1β, tumor necrosis factor-α, CXCL10/IP-10, epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), β2 microglobulin, cystatin C, myeloperoxidase, platelet-derived growth factor-AB/BB, CCL5/RANTES, soluble intercellular adhesion molecule-1, and soluble vascular cell adhesion molecule-1. A suspension array instrument (Bio-Plex 200, Bio-Rad) was used for cytokine data acquisition.

Prior to FACS analysis, individual cryovials of cells were rapidly thawed in a 37°C water bath (1–2 min agitation), resuspended in prewarmed animal component-free medium (Stem Cell Technologies) and seeded into separate wells of a six-well plate for 72 hours of incubation at 37°C. Cells were pooled, harvested by trypsinization with TrypLE™ Select, washed with culture medium and resuspended in autoMACS Rising Solution containing 0.5% BSA. The following antibodies were used for the isolation of CD81⁺ APC (Lin⁻: PDGFRα⁺: CD81⁺) from subcutaneous WAT in human: CD14-FITC (1:400, 301803, Biolegend), CD31-FITC (1:200, 303103, Biolegend), CD45-FITC (1:200, 304005, Biolegend), CD235a-FITC (1:500, 349103, Biolegend), PDGFRα (CD140a)-PerCP-Cy5.5 (1:200, 563575, BD Biosciences) and CD81-APC (1:500, 349510, Biolegend) in autoMACS Rising Solution containing 0.5% BSA in the dark at 4°C for 15 min. This sorting strategy was used to evaluate the correlations between CD81⁺ APC and clinical parameters. For intracellular staining, isolated SVF cells were stained with above antibodies without overnight culture, and immediately fixed by using Fixation/Permeabilization Solution Kit (BD Biosciences) as described protocol and stained with Ki67-PE antibody for 30 min (1:500, 350503, Biolegend). Cell population (%) was calculated as frequency of parent.

To characterize EVs, PFP samples were stained and acquired as previously described¹¹ using 14 different fluorochrome-conjugated antibodies in three separate panels, including CD235a-FITC, CD62P-APC, CD3-PerCP/Cy5.5, CD19-Alexa/700, CD28-FITC, CD16-V450, CD62L-APC, CD11b-PE/Cy7, CD66-PE (Biolegend), CD15-FITC (ExAlpha), CD152-APC, CD14-APC/Cy7, CD108a-PE, and CD41a-PerCP/Cy5.5 (BD Biosciences). Samples were washed with a 0.22 μm centrifugal filter (Millipore) at 500g for 5 minutes. EVs were then harvested from the top of the filter after washing, and data were acquired by an LSR II flow cytometer (BD Biosciences). Analysis of data was performed using FlowJo 7.6.5 software (Tree Star).