Anti cd146 pe

Phenotypical characterization of each cell line was performed by multicolor flow cytometry. Cells were harvested when 70–80% confluent, washed with PBS and incubated with PE- or FITC-conjugated antibodies for 1 h at 4 °C according to the manufacturer’s recommendation and then analyzed. Cells were characterized with the following antibodies: anti-CD56-FITC, anti-CD49b-FITC, anti-CD106-FITC, anti-CD197-FITC, and anti-CD146-PE (Beckman Coulter) (all from BD Pharmingen, Franklin Lakes, NJ, USA); anti-ICAM1-PE and anti-ICAM2-PE (both from Biolegend, USA); anti-alphaV-beta3-FITC (eBioscience^TM, San Diego, CA, USA). Isotype-matched non-reactive fluorochrome-conjugated antibodies were used as controls and quantitative analysis was performed using a Navios EX flow cytometer (Beckman Coulter, Brea, CA, USA) with software Navios (Beckman Coulter, CA, USA). In this study, at least 10,000 events were analyzed for each sample excluding non-viable cells based on forward scatter and side scatter parameters. The data are expressed as the ratio of mean fluorescence intensity (MFI) of each specific antibody and the relative isotype control. Values greater than 1 indicate expression of the specific marker. The above-mentioned surface markers were analyzed in separate experiments both in the original cell line and in the population of transmigrated cells.

The myogenic nature of purified cells was evaluated by flow cytometry analysis performed on CytoFLEX (Beckman Coulter) as previously described (Awaya et al., 2012 (link); Lapan and Gussoni, 2012 (link)); the following panel of antibodies was used to determine immunophenotype: anti-CD56 PC7 (Beckman Coulter, USA, A21692), anti-CD146 PE (Beckman Coulter, USA, A07483), anti-CD166 PE (Beckman Coulter, USA, A22361), anti-CD73 PE (BD Pharmingen, USA, 550257), anti-CD105 APC (R&D Systems, USA, FAB1097A-100), and anti-CD45 PC5 (Beckman Coulter, USA, A07785). Data were analyzed using the CytExpert 2.0 (Beckman Coulter). The phenotypic characteristics of the obtained cells are illustrated in the Supplementary Material, Figure S2A.

The immunophenotype of stem cells was evaluated by flow cytometry analysis performed on CytoFLEX (Beckman Coulter). Сells were resuspended in 100 μL of PBS containing 1% of bovine serum albumin (Sigma-Aldrich, Saint Louis, MO, USA) and incubated for 20 min at 20°C in the dark with the following monoclonal antibodies (Ab): anti-CD56 PC7 (Beckman Coulter, USA, A21692), anti-CD146 PE (Beckman Coulter, USA, A07483), anti-CD166 PE (Beckman Coulter, USA, A22361), anti-CD73 PE (BD Pharmingen, USA, 550257), anti-CD105 APC (R&D Systems, USA, FAB1097A-100), anti-CD45 PC5 (Beckman Coulter, USA, A07785), anti-PDGFRβ APC (BD Pharmingen, USA, FAB1263A), and anti-CD140a PE (BioLegend, USA, 323506). Data were analyzed using the CytExpert 2.0 (Beckman Coulter).