Anti cd133

Immunofluorescence staining in hDPCs was performed as previously described [37 (link)]. Briefly, hDPCs were treated with rhIGFBP-1 (100 ng/ml) for 2 days. The cells were fixed in 10% neutral buffered formalin solution at room temperature for 30 minutes, permeabilized in 2.5% Tween 20 (cat. no. 9005-64-5, Sigma-Aldrich) in PBS for 5 min, then incubated overnight at 4℃ with anti-ALP (1:500, cat. no. SC-15065, Santa Cruz), anti-β-catenin (1:500, cat. no. BD610154, BD Biosciences), anti-CD 133 (cat. no. NB120-16518, Novus Biologicals, CO, USA), anti-IGF-1 (cat. no. SC-9013 Santa Cruz), or anti-IGFBP-1 (cat. no. sc25257, Santa Cruz) primary antibody in blocking solution (1:1000). After several washes with PBS containing 0.5% Triton X-100 and 0.5% BSA, the hDPCs were incubated with anti-rabbit Cy2 (cat. no. 711-225-152, Jackson ImmunoResearch Inc., PA, USA) or anti-mouse (cat. no. 715-165-150; Jackson ImmunoResearch Inc.) labeled secondary antibodies for 1 h at RT. Following nuclear staining with DAPI dye (Sigma-Aldrich), the fluorescence signals were viewed using a Zeiss confocal laser microscope (LSM 700; Zeiss, Heidelberg, Germany).

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.