Anti cd166

BM aspirates were taken from the iliac crest of a healthy male donor after informed consent was provided (approved by the INHA University Medical School Institutional Review Board; IRB Number 10-51). Isolation of hcMSCs was carried out as previously described (21 (link)). Several cell surface antigens on the established hcMSC line, named KBHD502, were characterized by flow cytometry. The antibodies used for the analysis were anti-CD14, anti-CD29, anti-CD31, anti-CD34, anti-CD44, anti-CD73, anti-CD90, anti-CD105, anti-CD119, anti-CD133, anti-CD166, anti-HLA class I, anti-HLA-DR, anti-Stro-1, anti-c-Met, and anti-c-Kit (BD Biosciences Pharmingen, San Diego, CA, USA). The cells were analyzed in a FACSCalibur flow cytometer (BD Biosciences). Isotype-matched control antibodies were used as controls.

Bone marrow aspirates were collected from the iliac crest of a healthy male donor after obtaining informed consent (approved by Inha University Medical School Institutional Review Board). Isolation of the mesenchymal stromal cells and subsequent culturing was performed as previously described37 (link). The established mesenchymal stromal cell line was then analyzed for several stem cell markers using flow cytometry. The antibodies used for the analysis were as follows: anti-CD14, anti-CD29, anti-CD31, anti-CD34, anti-CD44, anti-CD73, anti-CD90, anti-CD105, anti-CD106, anti-CD166, anti-CXCR-4, anti-HLA-DR, anti-PODXL and anti-Oct-4 antibodies (BD Biosciences Pharmingen, San Diego, CA, USA). For the Oct-4 analysis, the cells were permeabilized with Triton X-100, and the cells were then analyzed by a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA). Isotype-matched control antibodies were used as controls.

Phenotypes of expanded MSC were analyzed upon staining with the following antibodies: allophycocyanin (APC)-labeled anti-CD34 (clone 581), anti-CD90 (clone 5E10), phycoerythrin (PE)-labeled anti-CD31 (clone WM59), anti-CD73 (clone AD2), anti-CD44 (clone G44-26), anti-CD29 (clone MAR4), fluorescein-isothiocyanate (FITC)-labeled anti-CD45 (clone 2D1) (all from BD Biosciences, San Jose, CA) and anti-CD105 (clone SN6, AbDSerotec, Raleigh, NC). For the analysis of CRC cells in coculture with stromal cells, the following antibodies were used: APC-labeled anti-EpCAM (clone EBA-1), FITC-labeled anti-CD90 and PE-labeled anti-CD44, anti-CD166 (all from BD Biosciences) or anti-CD133 (Miltenyi Biotec, Auburn, CA) or PE-labeled anti-TGF-β (clone 9016, R&D Systems). Propidium iodide (PI, 0.5 µg/mL) was added to all samples prior to analysis. Samples were analyzed by a dual laser BD FACS Calibur flow cytometer (BD Biosciences), following exclusion of dead cells based on PI incorporation. Analysis was performed using FlowJo software (Tree Star, Ashland, OR). For sorting of tumor cells and MSC from cocultures, cells were stained with APC-labeled anti-EpCAM and FITC-labeled anti-CD90. Dead cells were excluded based on DAPI incorporation. Cell sorting was performed using a BD Influx cell sorter (BD Biosciences). Purity of sorted cells was ≥98%.