Cd26 pe

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.

Cells were stained for 10 min at room temperature with the antibody against specific protein or mouse IgG2a isotype antibody. The cells were then washed with Dulbecco’s phosphate buffered saline (D-PBS; Corning, Manassas, VA, United States) and fixed with 1% paraformaldehyde. Protein expression was measured by flow cytometry on a MACSQuant Analyzer 10 (Miltenyi Biotec GmbH). At least 10,000 gated cells were used for the measurement of expression. Antibodies against the following proteins were used for flow cytometry analysis: CD26-PE (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), CD34-FITC, CD45-FITC, CD73-PE (BD Bioscience, San Diego, CA, United States), CD90-PE, CD105-PE (eBioscience, San Diego, CA, United States). To isolate cells based on their expression level of CD26, cells were incubated with CD26 antibody for 10 min at 4°C. Cells with a CD26 expression level above 85th percentile were sorted as CD26-positive (CD26⁺) and below 15th percentile were sorted as CD26-negative (CD26⁻). A FACSAria II (BD Bioscience) was used for cell sorting.

Following fluorochrome-conjugated antibodies were used for the evaluation of surface markers: CD44-PE, CD24-PE, CD26-PE, CD271-PE, CD133/2-PE (Miltenyi Biotec, Germany); CD166-PE (ALCAM; Immunotech, France); CD274 (PD-L1; Sony Biotechnology, USA); CD44v6-PE (RD Systems, USA); CD184 (CXCR4-PE; eBioscience, USA). Dead cells were excluded from the analysis based on DAPI staining. Cells were analyzed using BD FACSCanto™ II flow cytometer (Beckton Dickinson, USA) equipped with FacsDiva program. FCS Express software was used for the evaluation.