Anti cd105 pe

The bone marrow samples were obtained from the Second Affiliated Hospital of Nanchang University, China. Informed consent was obtained from all patients included in the study. MSC were isolated and cultured in DMEM/F12 (Gibco) medium containing 10% fetal bovine serum (FBS, Gibco), 2.0 mM glutamine, penicillin (100 U/mL), and streptomycin (100 U/mL) at 37°C in a humidified atmosphere containing 5% CO₂.
The immunophenotype of MSC was analyzed by cytofluorimetric analysis. Before experiments, the following monoclonal antibodies were used: anti-CD105-PE, anti-CD34-FITC, anti- CD45-FITC, and anti-CD90-PE (eBioscience, USA). BD FACSDiva flow cytometry (BD FACS Canto™ II, USA) was used. The adipogenic, osteogenic, and chondrogenic differentiation abilities of MSC were determined as previously described.

Anti-Sca1-PE, CD44-PE, CD73-PE, CD90-PE, CD34-PE, CD45-PE, CD4-PerCP, CD25-APC, CD3e, and CD28 antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD105-PE, Foxp3-PE, IL17-PE, and IFNγ-APC antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-FasL antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The phenotype of hESC-MSCs was characterized using flow cytometry and the following monoclonal antibodies: anti-CD31-APC (Miltenyi Biotec), anti-CD44-FITC (BD Pharmingen), anti-CD45-FITC (BD Pharmingen), anti-CD73-APC (Miltenyi Biotec), anti-CD90-FITC (BD Pharmingen), anti-CD105-PE (eBioscience), HLA-ABC-APC (BD Pharmingen), and HLA-DR-FITC (BD Pharmingen). Briefly, the cells were dissociated and suspended in FACS buffer (1x PBS/0.5% BSA) and nonspecific binding blocked with FcR blocking agent (Miltenyi Biotec) for 10 minutes at 4°C. For labeling cell surface antigens, the cells were incubated with the abovementioned fluorescent conjugated antibodies for 10 minutes at 4°C. After antibody labeling, data was acquired using Dako Cytomation CyAn ADP and analyzed using FlowJo v7.6.5 (Tree Star).

Cells were blocked with 50% rat serum and mouse Fc blocker (BD Bioscience) for 10 min, then stained for 30 min with fluorophore-conjugated antibodies, anti-Lin-FITC (eBioscience; 17A2/RA3-6B2/M1-70/TER-119/RB6-8C5, 1:25), anti-c-kit-PE (eBioscience; 104D2, 1:25), anti-CD105-PE (eBioscience, MJ7/18, 1:25), anti-CD11b-FITC (eBioscience; M1/70, 1:25), anti-CD34-PE (Invitrogen; MEC14.7, 1:25), anti-CD45-PE (eBioscience; 30-F11, 1:25), or their corresponding isotype control antibodies (eBioscience). For CXCR4 staining, primary monoclonal CXCR4 antibody (abcam; EPUMBR3, 1:25) and FITC- or APC-conjugated secondary antibody (goat-anti-rabbit IgG; BD Bioscience) were used. The cells were firstly gated for the intact cell population using forward scatter versus side scatter plots. All flow cytometry data were acquired on an LSRII (BD Biosciences, CA) and analyzed with FlowJo (Treestar, OR).

BM-MSCs of passages 3–5 or transfected BM-MSCs were harvested and incubated with anti-CD90-FITC (11-0909), anti-CD90-PE (12-0909), anti-CD105-PE (12-1057), anti-CD73-APC (17-0739), anti-CD45-FITC (11-9459), anti-CD45-PE (12-9459), anti-CD34-PE (12-0349), anti-CD19-APC (17-0199), and anti-CD11b-PE (12-0113, eBioscience) antibodies at 4°C for 30 minutes. Results were detected by using an FC 500 MCL Flow Cytometer (Beckman Coulter). Appropriate isotype-matched antibodies (eBioscience) were applied as controls.

MSCs were isolated from the femurs and tibias of mice. Briefly, the femurs and tibias were flushed with pre-cold phosphate-buffered saline (PBS) until they turned white. Thereafter, the cell suspension was passed through a 70-μm cullender (Falcon, Corning, NY) to obtain MSCs. The obtained MSCs were cultured for 3 days in 100-mm culture dishes with low-glucose Dulbecco’s modified Eagle’s medium (Hyclone, Waltham, MA) containing 10% fetal bovine serum and 1% 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Waltham, MA) in an incubator. The non-adherent cells were discarded, and MSCs were trypsinized to obtain plastic-adherent cells. MSCs in the fourth through tenth passages were used in the subsequent experiments. They were identified by fluorescence-activated cell sorting (FACS) using anti-CD29-FITC, anti-Sca-1-PE, anti-CD105-PE, anti-CD90-FITC, anti-CD45-FITC, anti-CD11b-PE, anti-CD34-PE, and anti-CD80-PE antibodies (eBioscience, Waltham, MA).
MSCs were treated with 1 nM TCDD (AccuStandard, New Haven, CT) for 24 h to activate AhR and harvested for the subsequent experiments. Both MSCs and AhR-activated MSCs were stimulated by TNF-α and IFN-γ (PeproTech, Rocky Hill, NJ) at a concentration of 2 μg/mL to induce immunosuppression in the co-culture experiments.