Cd34 fitc cd117 pe cd14 pc5 cd45 pc7

OB were seeded at 25 cm² flasks (Corning) coated with 0.02% gelatin (Sigma Aldrich, St. Louis, MO, USA) at a density of 400 × 10³ cells per flask. On day 4 the cells were removed and immunostained with the following panels of monoclonal antibodies: CD44-FITC/CD73-PE/CD90-PC5/CD105-PC7 and CD34-FITC/CD117-PE/CD14-PC5/CD45-PC7 (Beckman Coulter, Fullerton, CA). Analysis was performed using a flow cytometer according to standard protocols recommended by the manufacturer. The autofluorescence level was evaluated using an unstained control sample. The level of non-specific binding of antibodies was determined using isotypic controls (mouse immunoglobulins conjugated to FITC, PE, PC5, PC7). Gating of fluorescence events was carried out using the viability parameter. The viability was estimated by forward and side scattering along with 7-aminoactinomycin D staining. In each sample, at least 15,000 “targeted events” (events determined as viable cells) were analyzed.

Cell immunophenotyping was performed using a flow cytometer Navios (Beckman Coulter, Brea, CA, USA) equipped with 2 lasers (488 nm and 638 nm), 8 fluorescence detectors and the original standard set of light filters (Blue Laser: 525/40, 575/30, 620/30, 695/30, 755LP; Red Laser: 660/20, 725/20, 755 LP). The staining and detection were performed according to the standard protocols recommended by the manufacturer.
After the epithelial-mesenchymal transition, neural crest stem cells usually start to express a set of surface markers typical for mesenchymal stem cells (MSC). Therefore, the following monoclonal antibody panels were used to identify positive and negative surface markers typical for MSC [20 (link)]: CD44-FITC/CD73-PE/CD90-PC5/CD105-PC7 and CD34-FITC/CD117-PE/CD14-PC5/CD45-PC7 (Beckman Coulter, Brea, CA, USA). Cells were subjected to flow cytometry on the 2nd passage. Autofluorescence level was evaluated using an unstained control sample. The level of non-specific binding of antibodies was determined using isotypic controls (mouse immunoglobulins conjugated to FITC, PE, PC5, PC7). Gating of fluorescence events was carried out using the viability parameter. The viability was estimated by forward and side scattering along with 7-aminoactinomycin D staining. In each sample, at least 15,000 “targeted events” (events determined as viable cells) were analyzed.