Cd166 pe

Tumor cells were trypsinized and washed in 1 × PBS. The cells were then stained with the antibodies of choice: CD166-PE (Biolegend, 343904) or CD133–647 (Biolegend, 141216) or CD44 (Biolegend, 103047); in 1% BSA(Bovine Serum Albumin)-PBS. Cell cycle of monolayers or spheres of the cells were analyzed by fixing the cells in cold 75% ethanol. The analysis was carried out by the core facility of LCCC at Georgetown University. The annexin V (Biolegend, 640912) and propidium iodide (Biolegend, 421301) experiments were carried out following instruction from the manufacturer.

Digested cells were suspended in HBSS at a density of 1–5 × 10⁷ cells/mL and stained for 30 minutes on ice with THY-1-APC (BD Pharmingen) and LNGFR-PE (Miltenyi Biotec) antibodies for sorting. Cultured cells (at three and five passages) were harvested using cell-dissociation buffer (Gibco). Cells (1.0 × 10⁵) were suspended in ice-cold HBSS and stained for 30 minutes on ice with the monoclonal antibodies CD45-PE-Cy7 (Tonbo Biosciences), CD29-PE, CD31-PE-Cy7, CD44-PE, CD105-PE and CD166-PE (BioLegend) for cell surface analysis. Flow cytometric analysis and cell sorting were performed on a triple-laser Moflo system (Beckman Coulter) and data were analyzed using Flowjo software (Tree Star).

Isolated hO-MSCs were characterized using fluorescence-activated cell sorting (FACS). At least 100,000 isolated hO-MSCs were used for the analysis of each cell surface marker. Briefly, hO-MSCs were harvested and stained with the cell surface marker antibodies described below for 30 min at 4 °C in the dark and analyzed using a flow cytometer (FACS Canto II, Becton Dickinson and Company, Franklin Lakes, NJ, USA) at the Soonchunhyang Biomedical Research Core Facility of the Korea Basic Science Institute (KBSI). Antibodies were diluted 1:20 with FACS buffer containing 1% BSA in PBS and fluorescence-labeled cells were kept on ice during FACS analysis. MSC-positive markers CD29-PE (cat# 303,004, BioLegend, San Diego, CA, USA), CD44-FITC (cat# 103,022, BioLegend, San Diego, CA, USA), CD73-FITC (cat# 344,016, BioLegend, San Diego, CA, USA), CD105-PE (cat# 12–1057-42, eBioscience, San Diego, CA, USA), and CD166-PE (cat# 343,904, BioLegend, San Diego, CA, USA); and MSC-negative markers CD45-FITC (cat# 368,508, BioLegend, San Diego, CA, USA), CD34-FITC (cat# 343,517, BioLegend, San Diego, CA, USA), and CD31-PE (cat# 303,106, BioLegend, San Diego, CA, USA) were used for flow cytometry.

Approximately 10⁵ MSCs were incubated with 10 μl monoclonal antibody conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): CD105-FITC (Biolegend, ImTec, Antwerpen, Belgium), CD90-PE (Biolegend), CD73-PE (Biolegend) and CD166-PE (Biolegend). Unbound antibody was washed after 15 min with 3 ml PBS (Gibco). The cell pellet was resuspended in 0.5 ml PBS (Gibco). Samples were analyzed with the flow cytometer Macs Quant (Miltenyi Biotec, Bergisch Gladbach, Germany) with 10,000 events recorded for each condition. The morphology of cultured cells was evaluated using light microscopy.

DPSCs were treated with 0.25% trypsin/0.02% EDTA (Sigma-Aldrich) and centrifuged to generate a cell pellet. Cells were labelled with 10 μL FcR blocking solution (Miltenyi Biotec, Bergisch Gladbach, Germany) and various antibodies (see below, 10 μL per 1 × 10⁶ cells unless stated) in a total volume of 100 μL labelling buffer (PBS, 2 mM EDTA (Alfa Aesar, Heysham, UK) and 0.5% (w/v) BSA (Sigma-Aldrich)) for 20 minutes at room temperature in the dark. Following labelling, 900 μL labelling buffer was added to each sample before centrifugation and resuspension in 500 μL labelling buffer. Samples were then analysed using an LSRII flow cytometer (BD Biosciences, San Jose, CA) running FACSDiva 8.0 software (BD Biosciences), subsequent data analysis was performed using Kaluza 1.3 (Beckman Coulter, Inc., Miami, FL). Antibodies used were: CD29-Alexa Fluor 488 (5 μL per 1 × 10⁶ cells), CD34-FITC (5 μL per 1 × 10⁶ cells), CD44-FITC, CD73-PE (2 μL per 1 × 10⁶ cells), CD90-APC and CD166-PE (all Biolegend, San Diego, CA). All threshold values were obtained by analysing autofluorescence of non-labelled hDPSCs and reactivity of isotype matched controls. Events were gated based on forward and side scatter and colour compensation was performed.

Samples from four women were used in this step. The cells were evaluated according to the criteria for characterization of MSCs^{33 (link)}. Regarding adhesion to polystyrene and formation of fibroblast-like forming colonies, immunophenotypic¹⁵ characterization was done by flow cytometry by labeling the cells with the following antibodies: CD73-PECy7, CD90-FITC, CD105- FITC, CD19-PE Cy7, CD34-APC Cy7, CD45-APC, HLA-DR-PERCP Cy5.5, CD44-PERCP Cy5.5, CD49c-PE, CD151-APC, CD166-PE, CD117–APC, SSEA4-FITC, Oct4-PE (Biolegend, San Diego, CA), and NANOG-PERCP Cy5.5 (BD Pharmingen). The analysis was done in BD FACSCanto or BD FACSVerse and the results were analyzed using BD FACSDiva.
Analysis of differentiation into the three main mesenchymal lines was performed using adipogenesis, chondrogenesis, and osteogenesis StemPro differentiation kits (Gibco), according to the manufacturer's protocol. 0.5% oil Red O (Sigma-Aldrich), Alcian blue (Sigma-Aldrich), and Alizarin red (Sigma-Aldrich) were used respectively to observe adipogenesis, chondrogenesis, and osteogenesis.

Pre-conjugated antibodies CD49c-APC and CD166-PE were purchased from BioLegend, San Diego, CA, USA. SSEA4-PE, CD29-APC, CD54-PE, CD90-FITC, CD105-APC, CD106-APC were purchased from Miltenyi Biotec Inc., San Diego, CA, USA. Isotype IgG control antibodies were also purchased from Miltenyi Biotec Inc. Cells to be stained were washed 2 times with 5.0 mL of sterile HBSS and detached using 2.0 mL of TrypLE Express (Life Technologies, Grand Island, NY, USA). Cells were washed with DMEM supplemented with 10% FBS and spun down using a centrifuge set for 300xg. Cells were washed once again with 5.0 mL sterile 1x PBS and spun down at 300xg. Viable cell number was quantified using a hemacytometer and 0.4% Trypan blue solution (Life Technologies, Grand Island, NY, USA). For each sample to be stained, 1.0 × 10⁶ viable cells were resuspended in 100 µL of Flow buffer (1x PBS, pH 7.2, 0.5% bovine serum albumin and 2 mM EDTA). Pre-conjugated antibody (10 µL) was added to the resuspension, mixed and incubated for 10 min in the dark at 4 °C. Cells were washed 3 times with 1.0 mL of 1x PBS and resuspended in 500 mL of Flow buffer before single channel FACS analysis using an Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA). Control experiments for non-specific staining using mouse IgG were performed alongside all experiments.