Anti cd44 fitc

For identification of immunophenotypes, aging ASCs and DFAT cells were harvested at P4. Cells were trypsinized, and viability was assessed using 0.4% Trypan Blue (Gibco) and found to be greater than 95%. Then, cells were separated into tubes containing 5 × 10⁵ cells each. The following mouse monoclonal anti-human antibodies conjugated with fluorescein isothiocyanate-conjugated (FITC) and phycoerythrin (PE) were used: anti-CD29-PE, anti-CD31-PE, anti-CD73-PE, anti-CD90-PE, anti-CD105-PE, anti-CD106-PE, anti-CD146-PE (all from BD Biosciences, CA, USA): anti-CD34-FITC, and anti-CD44-FITC (Beckman coulter Inc.); and immunoglobulin G1 isotype control (BD Biosciences). Each aliquot was incubated in the dark at 4 °C for 20 min. Then, cell pellets were washed with PBS and resuspended in 0.1% bovine serum albumin (BSA)/PBS. Flow cytometric data were analyzed with Guava Express Plus version 5.3 software (Guava Technology).

PDLSCs from P3 were harvested by trypsinization and split approximately 5 × 10⁵ cells per tube. Mouse monoclonal anti-human antibodies conjugated with fluorescein isothiocyanate-conjugated (FITC) and phycoerythrin (PE) were performed as follow: anti-CD-90-PE, anti-CD105-PE, anti-CD106-PE, and isotype control using immunoglobulin G (all from BD Biosciences, San Jose, CA); anti-CD-34-FITC, and anti-CD-44-FITC (Beckman coulter). Each aliquot was incubated in the dark at 4 °C for 20 min. Cell pellets were washed with PBS and resuspended in 1 % BSA/PBS. Flow cytometric analysis was performed in triplicate and determined in quantitative data using Guava Express Plus version 5.3 software (Guava Technology).

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).