A07782

After the cell count was obtained, isolated cells were washed with DPBS containing 0.2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and 1 mM ethylenediaminetetraacetic acid (EDTA; Gibco). About 1.5 × 10⁵ cells were mixed in each tube with the following antibodies: hCD31–fluorescein isothiocyanate (FITC) (1:10; IM1431U, Beckman & Coulter, Brea, CA, USA), CD44–FITC (1:10; 555478, BD Biosciences, Franklin, NJ, USA), hCD45–FITC (1:10; A07782, Beckman & Coulter), hCD81–allophycocyanin (APC) (1:10; 551112, BD Biosciences), hCD90–APC (1:100; 559869, BD Biosciences), CD105–phycoerythrin (PE) (1:5; A07414, B76299, Beckman & Coulter), CD146–PE (1:5; 5050-PE100T, BioCytex, Marseilles, France), CD49-PE (1:5; 559596, BD Biosciences), and GD2 (1:10; 554272, BD Biosciences). The cells were incubated for 90 min at 4 °C and then washed with DPBS containing 0.2% BSA and 1 mM EDTA. GD2 was recognized by incubating the cells with the secondary antibody FITC-conjugated anti-mouse IgG (1:20; 349031, BD Biosciences) for 10 min at 4 °C. Fluoroprobe-labeled mouse IgG1 antibody (1:10; Beckman & Coulter) was used as a negative control. Stained cells were analyzed using a FACSVerse™ cell sorter (BD Biosciences) (Supplementary Fig. 2).

Flow cytometry was used to characterise PBMCs for CD34, CD45, CD90 and CD105 cell surface markers using IM1839U, A07414, IM1870 and A07782 (Beckman Coulter) together with MSC antibody panel SC017 from R&D Systems (Stro-1, CD44, CD90, CD106, CD105, CD146 and CD166) according to the manufacturer’s instructions. Cells were analysed using a Beckman Coulter Cytomics FC500 flow cytometer instrument and the data was assessed with Kaluza Analysis Software. Positivity for each antibody was defined as the level of fluorescence >99% of the isotype-matched control.
Antibody-conjugated paramagnetic microbeads were used for a cell selection; CD14 Microbeads (130 050 200), CD105 Microbeads (130 051 200) and Monocyte isolation kit II (130 091 183) from Miltenyi Biotec and VersaLyse red blood cell lysing solution (A09777, Beckman Coulter) according to the manufacturer’s instructions. A heterogeneous mixture of peripheral blood mononucleated cells 2.0 x 10⁶/ml in PBS was discriminated using a FACSAria III (BD Biosciences, US) analyser according to light scatter signals FSC and SSC into monocytes, lymphocytes and granulocytes. Cell populations were analysed after normoxic and hypoxic culture conditions.

Flow cytometry was used to characterize CD90 and CD105 cell surface markers using CD90-FITC Mouse IgG (IM1839U) and CD105-PE Mouse IgG (A07414, Beckman Coulter). Additionally, CD34 (IM1870) and CD45 (A07782, Beckman Coulter) were used for negative selection. Fluorochrome-conjugated antibodies were incubated in 20 μL/2.0 × 10⁵ cell suspension in full media or PBS for 20 minutes at room temperature protected from light. Finally the cells were washed three times with PBS solution, resuspended into IsoFlow Sheath Fluid (8546859, Beckman Coulter), and the cell fluorescence was measured using Beckman Coulter Cytomics FC500 instrument. The data was assessed with Kaluza analysis software. Positivity for each antibody was defined as the level of fluorescence >99% of the isotype-matched control antibodies.