C met apc

Cells were incubated with antibodies in 200 μl PBS containing 2% FBS (wash buffer) at the dilutions suggested by suppliers for 30 min on ice. Then the cells were washed in wash buffer and analyzed and sorted using a BD FACSAria Fusion cell sorter (BD Biosciences). The following antibodies were used: CD29-PE (clone TS2/16, eBioscience), CD45-PE (clone HI30, BD Pharmingen), CD56-FITC (clone B159, BD Pharmingen), CD57(HNK-1)-PE (clone TB03, Miltenyi Biotec), CD82-APC (clone REA211, Miltenyi Biotec), ERBB3-APC (clone REA508, Miltenyi Biotec), CD105-FITC (clone 266, BD Pharmingen), CD108-PE (clone KS-2, BD Pharmingen), CD140a (PDGFRα)–PE (clone αR1, BD Pharmingen), CD146/MCAM -PE (clone P1H12, BD Pharmingen), CD271-PE (clone C40-1457, BD Pharmingen), CD271-BD Horizon BB515 (BD), CD318-APC (clone REA194, Miltenyi Biotec), CD325/N-cadherin-PE (clone 8C11, BioLegend), C-MET-APC (clone 95106, R&D systems), SSEA4-PE (clone MC813-70, BD Pharmingen), TRA-1-60-Alexa 488 (clone TRA-1-60, BD Pharmingen), and TRA-1-81-Alexa647 (clone TRA-1-81, BD Pharmingen). After sorting, cells were plated on collagen-coated 12- or 48-well plates and cultured up to 10 d.

For the evaluation of CSCs-like cell surface markers, the following fluorochrome-conjugated antibodies were used: CD24-PE, CD26-PE, CD44-PE/CD44-APC, CD133-PE/CD133-APC, CD271-PE, EpCAM-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD166-PE (ALCAM, Immunotech, France), and cMET-APC (R&D Systems, Abingdon, UK). Dead cells were excluded from the analysis based on DAPI staining. Cells were analyzed by BD FACSCanto™ II flow cytometer (Beckton Dickinson, USA) equipped with FacsDiva program. FCS Express software was used for the evaluation.