Cd36 apc

Each assay contained a unique combination of the following mouse anti-human monoclonal antibodies: CD73-FITC, CD90-APC, CD105-PE (BioLegend, San Diego, CA, USA), CD34-PE, CD36-APC, CD146-PE (Miltenyi BioTech, Bergisch, Germany), CD61-PE (Beckman Coulter Inc., Pasadena, CA, USA), CD15-FITC, and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA). All antibodies were titrated and used at a 50 ng/assay concentration. Isotype controls and specific mAbs were employed at the same final concentrations. Routinely, 50,000 hASCs (in 100 µL FACS buffer) were mixed with the appropriated antibody combination and incubated for 15 min at RT in the dark. Finally, the sample was diluted with 100 µL FACS buffer before the acquisition, according to our flow cytometer procedure (see Section 2.3.3).

Freshly isolated cells of the SVF were stained for analytical flow cytometry as described by Panella et al. [19 (link)]. Briefly, the cells were incubated 20 min with the following monoclonal mouse anti-human antibodies and fluorescent dyes: CD34-BV650, CD45-PC7, CD73-FITC (BioLegend, San Diego, CA, USA), CD146-PE, CD36-APC (Miltenyi BioTech, Bergisch, Germany), 7-amino-actinomycin D (7-AAD) (Becton Dickinson, Franklin Lakes, NJ, USA), and Syto40 (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). After that, the erythrocytes were lysed with 200 μL of VersaLyse solution (Beckman Coulter Inc., Pasadena, CA, USA). ASCs are described as CD45-, CD146-, CD36-, CD34+, and CD73+ cell populations. Samples were acquired according to our flow cytometer procedure. More details about this protocol can be found in the Supplementary Materials Table S1, which accompanies and complements this study.