Facs navio fc

Immunophenotypic characterization of isolated AECs and WJ-MSCs was performed using flow cytometry. Cells were fixed for 10 min at room temperature using Intraprep Kit (Beckman–Coulter Inc., Brea, CA, USA) and washed twice with PBS. Cells were incubated for 30 min at 4 °C with conjugated primary antibodies (1 μg/mL) specific for epithelial (anti-pan Cytokeratin (Pan-Ck)-PE, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mesenchymal (anti-CD44-PE, anti-CD73-PECY7, anti-CD90-FITC, anti-CD105-PECY7, Beckman-Coulter Inc., Brea, CA, USA), hematopoietic (anti-CD14-PE, anti-CD34-PerCP, anti-CD45-FITC, Beckman-Coulter Inc., Brea, CA, USA), and stem cell (anti-state-specific embryonic antigen-4 (SSEA4) APC, Miltenyi Biotech, Germany) markers. For the analysis of Pan-Ck, we used the Pan-Ck Type I/II Antibody Cocktail (MA5-13156, Thermo Scientific, Waltham, MA, USA) and anti-mouse–Alexa Fluor 488 (A11001, Thermo Scientific, Waltham, MA, USA) as a secondary antibody. After incubation, cells were washed with PBS and analyzed using the FACS Navio FC (Beckman-Coulter Inc., Brea, CA, USA) cytometer and the Kaluza FC C 1.2 Analysis software (Beckman-Coulter Inc., Brea, CA, USA).

Immunophenotypic characterization of hAECs was assessed after isolation by flow cytometry. hAECs were fixed for 10 min at room temperature using Intraprep Kit (Beckman–Coulter Inc., Brea, CA, USA) and washed twice with PBS. Cells were incubated for 30 min at 4 °C with conjugated primary antibodies (1 μg/mL) specific for epithelial (anti-panCytokeratin (Pan-Ck)-PE, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mesenchymal (anti-CD44-FITC, anti-CD73-PE, anti-CD90-PC5, anti-CD105-PE, Beckman-Coulter Inc., Brea, CA, USA), and hematopoietic (anti-CD34-FITC, anti-CD45-APC, Beckman-Coulter Inc., Brea, CA, USA) markers. For the analysis of Pan-Ck during culture passages, we used the Cytokeratin Pan Type I/II Antibody Cocktail (MA5-13156, Thermo Scientific, Waltham, MA, USA) and Alexa Fluor 488 (A11001, Thermo Scientific, Waltham, MA, USA) as secondary antibody. After incubation, cells were washed with PBS and analyzed using the FACS Navio FC (Beckman-Coulter Inc., Brea, CA, USA) cytometer and the Kaluza FC Analysis software (Beckman-Coulter Inc., Brea, CA, USA).