Anti cd144

Phenotypic characterization of ECFCs was performed using a BD FACSCanto II Flow Cytometer (BD Biosciences, Breda, the Netherlands). Cells were detached using accutase and checked for the following endothelial makers: anti‐hVEGFR2‐PE (R&D Minneapolis, MN), anti‐hVE‐Cadherin‐PE (R&D), anti‐CD31‐PE (R&D), anti‐CD90‐PE (R&D), anti‐CD105‐PE (R&D), anti‐CD34‐FITC (BD), anti‐CD90 AF647 (Biolegend), and anti‐CD133‐PE (Miltenyi, Bergisch Gladbach, Germany), as well as absence of haematopoietic/myeloid marker expression with anti‐CD45‐PE (BD) and anti‐CD14‐PE (Biolegend, San Diego, CA).
Additional characterization was performed by immunofluorescent staining. Cells were grown until confluency in chamber slides (Thermo Fisher, Landsmeer, the Netherlands), fixed with 4% formaldehyde and permeabilized with 0.1% Triton X‐100 where appropriate. Anti‐CD144 (R&D), anti‐CD31 (R&D), and anti‐von Willebrand Factor (vWF, Sigma) primary antibodies were used, secondary staining was performed with anti‐Mouse AF555 and anti‐rabbit AF488 secondary antibodies, and nuclei were visualized with 4′,6‐diamidino‐2‐phenylindole (DAPI). Images were taken with a Zeiss LSM700 Confocal Microscope. Fluorescent‐activated cell sorting (FACS) profiling was performed for one ECFC donor (Figure S2).

Cultures of PF ADSCs and BC ADSCs at different passages (lower than passage 4) were phenotypically characterized following reference guidelines [32 (link), 33 (link)]. ADSCs obtained from PF- or BC-bearing patients were detached with 0.05% trypsin/EDTA (Thermo Fisher), washed with PBS, and 100000 cells were resuspended in 250 μL of PBS without Ca²⁺ and Mg⁺ (Euroclone, Pero, Italy) and incubated with antibodies directed against specific surface markers. Cells were incubated on ice for 30 minutes with antibodies anti-CD44 (BD Biosciences, San Jose, CA), anti-CD90 (Millipore, Massachusetts, USA), anti-CD34 (Miltenyi Biotec, Calderara di Reno, BO, Italy), anti-CD45 (BD Biosciences), anti-CD146 (Biocytex, USA), anti-CD31 (Miltenyi Biotec), anti-CD56 (Miltenyi Biotec), anti-CD105 (Serotec, Bio-Rad, Segrate, MI, Italy), anti-CD144 (R&D Systems, Minneapolis, MN, USA), anti-CD166 (BD Biosciences), anti-CD133/2 (Miltenyi Biotec), anti-CD73 (BD Biosciences), and anti-vascular endothelial growth factor 2 (VEGFR2; R&D Systems). Cells were pelleted, washed, and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes. Fluorescence-activated cell sorting (FACS) analysis was performed on a FACSVerse flow cytometer (BD Biosciences), equipped with the Cell Sweet software for data analysis.

Cultures of mechanically activated hADSCs at different passages were phenotypically characterized by Fluorescence-Activated Cell Sorting (FACS). As previously reported by us for hADScs obtained from lipoaspirate [36 (link)], after trypsinization, cells were washed with PBS and 1 × 105 cells were re-suspended in 250 uL of PBS w/o Ca²⁺ and Mg⁺ (Euroclone, Pero, Italy) and incubated with antibodies directed against specific surface markers. The following antibodies were used: anti-CD44 (BD Biosciences, Milano, Italy), anti-CD90 (Merck Millipore, Vimodrone, Milano, Italy), anti-CD34 (Mylteni Biotec, Bologna, Italy), anti-CD45 (BD Biosciences), anti-CD146 (Biocytex, Marseille, France), anti-CD31 (Mylteni Biotec), anti-CD56 (Mylteni Biotec), anti-CD105 (Biorad, Milano, Italy), anti-CD144 (R&D System, Minneapolis, MN, USA), anti-CD166 (BD Biosciences), anti-CD133/2 (Mylteni Biotec), anti-CD73 (BD Biosciences), and anti-VEGFR2 (R&D System). Samples were fixed with paraformaldehyde 4% and then studied by flow cytometry (FACS Vantage, BD Bioscience, Milano, Italy) using a specific software (CellQuest Pro, BD Bioscience). Results were expressed as mean ± SD.