(CD14), SJ25C1 (CD19), MAR4 (CD29), 8G12 (CD34), 515 (CD44), 2D1 (CD45), IA10 (CD55), p282 (CD59), AD2 (CD73), 5E10 (CD90), SN6 (CD105), 104D2 (CD117), and L243 (HLA-DR). All of these monoclonal antibodies were obtained from BD Biosciences (San Jose, CA), except for SN6 from Invitrogen (Carlsbad, CA). Cells were resuspended in a total number of 2 × 105 in 50 μl of phosphate-buffered saline (PBS) supplemented with 4% FBS, then incubated with 20 μl of monoclonal antibodies, except for 5E10 (2 μl) and SN6 (5 μl), for 45 min at 4°C, and the conjugated cells fixed with 1 ml of 4% paraformaldehyde solution (Wako, Osaka, Japan). Flow cytometric analysis was performed with Cell Quest software and the FACSCalibur device (BD Biosciences) to examine 20,000 events. In vitro differentiation toward AZD7762 manufacturer adipocytes, chondrocytes, and osteocytes To induce adipogenesis and osteogenesis, 1 × 103 cells were cultured in 500 μl of medium in a four-well chamber slide. Three days after propagation, the culture medium was replaced with 500 μl of StemPro adipogenesis or osteogenesis differentiation medium (Gibco) containing 5 μg/ml of gentamicin. Chondrogenesis was induced with a micromass culture system [19, 20], in which 5 × 102 of the cells were resuspended in 10 μl of culture medium and applied to
the center of a culture well. A Selleck Bioactive Compound Library 96-well find more culture plate was used in our study. Two hours after propagation, 100 μl of StemPro chondrogenesis differentiation medium containing 5 μg/ml of gentamicin was added. The differentiation medium was replaced twice a week. Mixed lymphocyte culture assay PBMCs were separated from the heparinized peripheral blood of a healthy donor by means of Ficoll-Paque density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). CD3+ T-cells were purified from PBMCs by magnetic-activated cell sorting (MACS) positive selection (Miltenyi Biotec, Auburn, CA) and
1 × 106 of these cells were cultured for 48 h in a 96-well culture plate in the presence of 12.5 μg/ml of phytohemagglutinin (Wako) with or without irradiated (25 Gy) HPB-AML-I and UCBTERT-21 (0, 1 × 103, 1 × 104, and 1 × 105 cells/well) cells. From each culture well, 100 μl of cell suspension was pulsed with 10 μl of Cell Counting Kit-8 solution (Dojindo, Methamphetamine Tokyo, Japan) at 37°C for 4 h. The optical density at 450 nm was measured to determine cell viability in each of the culture wells. Results HPB-AML-I shows plastic adherence, negative myeloperoxidase expression, and complex chromosomal abnormalities Inverted microscopic examination (Figure 1A) and May Grünwald-Giemsa staining (Figure 1B) of HPB-AML-I cells revealed that this cell line is composed of round-polygonal and spindle-like cells. Unlike the round-polygonal cells, HPB-AML-I cells with the spindle-like morphology attached to plastic surfaces.