Cd166 pe

Cells were harvested after detachment with TrypLE Select, washed in PBS (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 30 min with the following monoclonal antibodies: CD105-FITC (Ancell corporation, Bayport, NY, USA), CD73-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD146-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD166-PE (BD Biosciences, Erembodegem, Belgium), CD45-PC7 (BD Biosciences, Erembodegem, Belgium), HLA-ABC- PC5 (BioLegend, San Diego, CA, USA), HLA-DR-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD47-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD200-PC7 (BD). After washing with PBS, the cells were fixed with 8% formaldehyde. Data were acquired and analyzed on a MacsQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).

100 μl DPSCs at a concentration of 1 × 10⁶ cells/ml were stained by 5 μl of each of the following human antibodies: CD45-PE, CD73-PE, CD90-APC, and CD166-PE (BD, USA). The samples were incubated at 37 °C for 30 min, centrifuged, washed twice with PBS, and examined by flow cytometry (BD, USA).

BMMSCs were isolated, cultured and induced into osteoblast. The procedure was detailed in our previous studies [32 (link), 33 (link)]. The immunophenotype of BMMSCs were evaluated at P3 (the third passage). After 15 min incubation with the antibodies as follow: CD105-FITC, CD73-FITC, CD29-FITC, CD90-FITC, CD166-PE, CD34-PE, CD45-FITC, CD80-FITC, CD86-FITC (BD Biosciences Pharmingen, San Diego, California, USA), CD44-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), BMMSCs were analyzed by FACS Calibur system (BD, Mountain View, CA, USA) using Cell Quest software.

For all 30 strains between the 4th and the 5th passage, immunophenotyping was performed by flow cytometry in a FACSCalibur flow cytometer (BD, Becton Dickinson Franklin Lakes, NJ) and analyzed in the CellQuest program (BD, Becton Dickinson Franklin Lakes, NJ). Immunophenotyping allows the characterization of cells at different stages of development through the use of fluorescent monoclonal antibodies against surface markers (antigens).
Cells obtained from cell cultures at a concentration of 1 × 10⁶ cells/100 μl were labeled with the following monoclonal antibodies: CD29-PE, CD31-FITC, CD34-FITC, CD45-PE, CD73-FITC, CD90-FITC, CD105-PE, CD166-PE, IgG-FITC, and IgG-PE isotypes (BD Biosciences, Becton Dickinson, Franklin Lakes, NJ) for 15 minutes at room temperature in the dark. Five hundred microliters of PBS was then added with 3% FBS and incubated for 15 minutes at room temperature in the dark. First, unstained cells were analyzed, and from that analysis, specific isotypes for each antibody were used for staining, with monoclonal antibodies as a negative control for the reaction, and were measured the minimum 5 × 10⁵ events.

CmPC cultured in the indicated conditions were detached using a non-enzymatic method, cells were resuspended in PBS containing 0.1% BSA (Gibco) and 2 mM EDTA (Gibco) and incubated in the dark for 15 min with suitable combinations of the monoclonal antibodies or isotype-matched control monoclonal antibodies: CD29-PE, CD44-PE, CD73-PE, CD166-PE, CXCR4 APC (BD Pharmingen). Samples were then washed with 1 ml of washing buffer and centrifuged for 10 min at 400 × g at 4 °C to remove unbound antibodies. Cells were resuspended in 250 μl of washing buffer and analyzed.
For Figure 5c, CmPC were incubated with Brilliant Violet 421(BV421)-conjugated anti-CXCR4 antibody (Biolegend, San Diego CA) used at 1 μl in 30 μl final dilution for 15 min at RT. The CXCR4 signal (450 nm wavelenght) was plotted against an empty channel of the FACSAria cell sorter (Beckton Dickinson, Franklin Lakes, NJ, USA) receiving autofluorescence detected at 530 nm wavelenght (AF 530) to set up proper positivity gates. Numbers indicate percentages of CXCR4-positive cells for the indicated conditions. Flow cytometry data was analyzed by FlowJo software ver 9.9.5 (Treestar, Ashland, OR, USA).