Anti cd90 pe

Umbilical cord blood was collected in several hospitals using Stemcare/CB collect blood bag system (Fresenius Kabi Norge AS) containing citrate phosphate dextrose (CPD) as an anticoagulant. Approval for collection was obtained from the Medical Ethical Committee of the Erasmus University Medical Centre (MEC-2009–410) and written informed consent from the mother was obtained prior to donation of the cord blood. Within 48 hours after collection, mononuclear cells were isolated using ficoll (Lymphoprep, Fresenius Kabi Norge AS). CD34⁺ cells were isolated with double positive immunomagnetic selection using Magnetic Activated Cell Sorting (MACS) technology according instructions of the manufacturer (Miltenyi Biotech GmBH, Bergisch Gladbach, Germany). MACS-selected CD34⁺ cells were either used directly in experiments or stained with anti-Lin-FITC, anti-CD38-PerCP-Cy5.5, anti-CD90-PE (all from eBioscience, Vienna, Austria), anti-CD34-PE-Cy7, anti-CD45RA-APC-H7 (both from BD Biosciences, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA) after which viable DAPI^-Lin^-CD34⁺CD38^lowCD45RA^lowCD90⁺-cells, highly enriched for hematopoietic stem cells (HSC)[30 (link)], were sorted using BD FACSAria Cell Sorting System (BD Biosciences, San Jose, CA, USA).

Phenotype analysis was performed as described previously [77 (link)]. Passage 2 cADSC were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences, Franklin Lake, NJ, USA; 2 × 10⁵ cells/tube), washed with FACS buffer (PBS containing 2% FBS), followed by blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated on ice for 20 min with the following fluorescein (FITC)- or phycoerythrin (PE)-conjugated antibodies: anti-CD14-FITC (BD Pharmingen, San Diego, CA, USA), anti-CD29-PE (BioLegend, San Diego, CA, USA), anti-CD34-PE (R&D Systems, Minneapolis, MN, USA), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience, San Diego, CA, USA), and anti-CD90-PE (eBioscience) or their respective isotype controls. The cells were washed twice with FACS buffer and resuspended in 500 μL FACS buffer. Fluorescence was evaluated by flow cytometry in a CytoFLEX instrument (Beckman Coulter, Brea, CA, USA). The data were analyzed using CytExpert ver2.0 analysis software.

Passage 2 AT-MSCs were analyzed by flow cytometry. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 10⁵ cells/tube), washed with FACS buffer (PBS containing 2% FBS), blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated with the following fluorescein- (FITC-) or phycoerythrin- (PE-) conjugated antibodies: anti-CD14-FITC (BD PharMingen), anti-CD29-PE (BioLegend), anti-CD34-PE (R&D Systems), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience), and anti-CD90-PE (eBioscience) or their respective isotype controls listed in Table 1. The cells were washed twice with FACS buffer and resuspended in 500 μl FACS buffer. Fluorescence was evaluated by flow cytometry in a FACSCalibur instrument (BD Biosciences). Data were analyzed using WinMDI 2.9 analysis software.

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.

The cell surface markers were analyzed with flow cytometry. The primary ADSCs at passage 3 (n = 3), and the ADSC-K4DT (n = 3) and ADSC-K4D (n = 3) cells were analyzed at low (approximately PDL 20), intermediate (approximately PDL 50), and high (approximately PDL 100) PDs. The cells were washed with FACS buffer (PBS containing 2% FBS), and the Fc receptors were blocked with canine Fc receptor binding inhibitor (Thermo Fisher Scientific). Then, the cells were incubated with the following phycoerythrin (PE)-conjugated antibodies: anti-CD29-PE (clone: TS2/16; BioLegend, San Diego, CA, USA), anti-CD34-PE (clone: 1H6; R&D Systems, Minneapolis, MN, USA), anti-CD44-PE (clone: IM7; BioLegend), anti-CD45-PE (clone: YKIX716.13; eBioscience, San Diego, CA, USA), anti-CD90-PE (clone: YKIX337.217: eBioscience), and anti-HLA-DR-PE (clone: G46-6: BD Biosciences, Franklin Lake, NJ, USA). All the cells at each passage were examined in triplicate.