Anti cd166 pe

To confirm their identities as MSCs, both SHED and OOMDSCs were characterized by flow cytometry for the expression of typical surface markers of MSCs (CD29, CD44, CD90, CD73, CD105, and CD106) and endothelial (CD31) and hematopoietic (C34) cells. All cells were incubated with antibodies (conjugated with fluorochromes) that had the ability to bind specifically to intracellular and cell surface proteins to compare and characterize the cells according to the expression of specific antigens. A total of 1 × 10⁶ cells obtained from cell cultures and diluted in 100 μL of 1XPBS⁻ were transferred to flow cytometry tubes and incubated with the following monoclonal antibodies for 15 minutes at room temperature in the dark for staining: anti-CD29-PE, anti-CD44-PE, anti-CD73-FITC, anti-CD90-FITC, anti-CD105-PE, anti-CD166-PE, anti-CD34-FITC, and anti-CD31-FITC (BD Bioscience, Becton Dickinson Franklin Lakes, NJ). The samples were then washed with 1XPBS⁻, resuspended in 500 μL of 1XPBS⁻, run on a FACSCalibur (BD, Becton Dickinson, Franklin Lakes, NJ) flow cytometer, and subsequently analyzed using FlowJo software (TreeStar Inc.). A sample of unstained cells was prepared for each experiment to eliminate the influence of any nonspecific staining and innate autofluorescence of the cells. As a negative control for the reactions, an isotype control was used for each antibody.

ADSCs of all donors were expanded separately to passage four, pooled, and examined once for surface marker expression using flow cytometry as a pool of six donors. The following monoclonal antibodies conjugated to fluorochromes were used: anti-CD11b-APC, anti-CD13-APC, anti-CD29-PE, anti-CD31-FITC, anti-CD34-FITC, anti-CD44-APC, anti-CD45-FITC, anti-CD63-FITC, anti-CD73-PE, anti-CD90-APC, anti-CD105-FITC, anti-CD106-APC, anti-CD-166-PE, and anti-CD235a (all from Becton Dickinson, Heidelberg, Germany). Isotype antibodies were included for all fluorochromes.
Cells were detached with 0.25% trypsin-EDTA, washed in FACS buffer (1% FCS, 0.1% NaN₃ in PBS), incubated with directly conjugated monoclonal antibodies (5 μl/100,000 cells) in FACS buffer for 30 min on ice, washed twice with FACS buffer, and fixed with 1% paraformaldehyde/PBS. Cells were analyzed using a FACSCanto flow cytometry system (Becton Dickinson). Data acquisition was performed with Diva software (Becton Dickinson) and data were analyzed using FCS express V3 (De Novo Software).

ADSCs expanded to passage four were examined for surface marker expression using flow cytometry. The following monoclonal antibodies conjugated to fluorochromes were used: anti-CD13-APC, anti-CD29-PE, anti-CD31-FITC, anti-CD34-FITC, anti-CD44-APC, anti-CD45-FITC, anti-CD49a-PE, anti-CD63-FITC,-anti-CD73-PE, anti-CD90-APC, anti-CD105-FITC, anti-CD106-APC and anti-CD-166-PE (all from Becton Dickinson, Heidelberg, Germany). Isotype antibodies were included for all fluorochromes.
Cells were detached with 0.25% trypsin-EDTA, incubated with directly conjugated MAbs in FACS-buffer (1% FCS, 0.1% NaN₃ in PBS) for 30 minutes on ice, washed twice with FACS buffer, and fixed with 1% paraformaldehyde/PBS. Cells were analyzed using a FACSCanto flow cytometry system (Becton Dickinson). Data acquisition was performed with Diva software (Becton Dickinson) and data were analyzed using FCS express V3 (De Novo Software).