Isolation and Characterization of Endothelial Colony Forming Cells

Endothelial colony forming cells (ECFCs) were isolated from white adipose tissue, as described [28 (link)]. Briefly, ECFCs were cultured in 75 cm² flasks, treated with 1% gelatin (G1890, Sigma, Steinheim, Germany) and incubated in 20% FBS/ EBM-2 media (CC-3156, Lonza, Basel, Switzerland) containing 1% penicillin/streptomycin (P/S) and the necessary growth factors (VEGF, hFGF-B, hEGF and R3-IGF-1), ascorbic acid and heparin, without hydrocortisone (CC-4147, Lonza). Cells were grown until confluence (90%) was reached. Experiments were performed on passages 6–7.
Cell identity was confirmed by testing cloning-forming ability, as described [2 (link)], and also by flow cytometry, analyzing several specific antibodies against CD31, CD14, CD90, CD34, CD45, CD73, CD133, CD309 and CD146 (Supplementary Figure S3). An isotype IgG1 antibody was used for negative control. The full list of antibodies is shown in Supplementary Table S1. Fluorescence was measured using CytoFLEX cytometer (Beckam Coulter, West Sacramento, CA, USA) and CytExpert software. Finally, data were analyzed with FlowJo v10.4 software.