Fitc conjugated cd29

Antibodies were purchased from the following sources: PE conjugated CD105, FITC conjugated CD29, APC conjugated CD31, PerCP-Cy5.5 conjugated CD45, and PE-Cy7 conjugated CD34 were from eBioscience. p63, AR, CK8, CK5, PSA and vimentin antibodies were from Santa Cruz Biotechnology. Testosterone and collagenase IV were from Sigma and human nuclei antibody was from Millipore.

P3 MenSCs-DGC (n=5) and MenSCs-RLB (n=5) were used for the immunophenotyping analysis. Mouse anti-human monoclonal antibodies: FITC-conjugated CD29 (Cat#: 11-0299-42, dilution ratio 1:20, eBioscience), CD44 (Cat#: 11-0441-82, dilution ratio 1:20, eBioscience), CD73 (Cat#: 11-0739-42, dilution ratio 1:20, eBioscience), CD90 (Cat#: 11-0909-42, dilution ratio 1:20, eBioscience), HLA-ABC (Cat#: 11-9983-42, dilution ratio 1:20, eBioscience), HLA-DR (Cat#: 11-9956-42, dilution ratio 1:20, eBioscience), CD34 (Cat#: 11-0349-42, dilution ratio 1:20, eBioscience), CD45 (Cat#: 11-9459-42, dilution ratio 1:20, eBioscience) and PE-conjugated CD105 (Cat#: 12-1057-42, dilution ratio 1:20, eBioscience) were used. As negative controls, isotype PE (Cat#: 12-4714-42, dilution ratio 1:20, eBioscience) and FITC-conjugated IgG (Cat#: 31505, dilution ratio 1:20, eBioscience) were used. The cell suspensions (1×10⁶ cells) were washed twice with PBS and incubated with the monoclonal antibodies at 4°C in the dark for 30 min. After washing with PBS, the samples were resuspended and analyzed using a Cytomics FC 500 MPL cytometer (Beckman Coulter).

MenSCs harvested from different passages were used for immunophenotyping analysis. Mouse anti‐human monoclonal antibodies: FITC‐conjugated CD29, CD73, CD90, HLA‐ABC, HLA‐DR, CD34 and CD45 and PE‐conjugated CD105, and rat anti‐human monoclonal antibodies: FITC‐conjugated CD44 (eBiosciences, San Diego, CA, USA) were used. As a control, isotype PE and FITC‐conjugated IgG were used. The cell suspension (1 × 10⁶ cells) was washed by PBS for twice and incubated with monoclonal antibodies at 4°C in the dark for 30 min. After washing with PBS, the samples were analysed by Cytomics FC 500 MPL cytometer (Beckman Coulter, Brea, CA, USA).