Anti cd90

BMSCs cells were isolated from Wistar rats (weighted 80–100 g; specific pathogen-free grade; Vital River Laboratories, Beijing, China) and cultured as previously described (Hussain et al., 2016 (link)). Rats were anesthetized by intraperitoneal (IP) injection of chloral hydrate (350 mg/kg), and the femur and tibia of rats were excised and bone marrow cells were slowly flushed out from the marrow cavity using DMEM medium (Gibco, Grand Island, NY, USA). Cell was cultured with DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA). Non-adherent cells were removed after 48 h. Approximately 14 days later, the adherent stromal cells become confluent and were designated passage 0 (P0). The BMSCs were expanded and passaged to P3, at which point they were harvested and employed in the present study. Animal experiments performed in this study were all approved by the Animal Ethics Committee of Southern Medical University. Precautions were taken to minimize suffering and the number of animals used in each experiment.
BMSCs were identified by the expression of specific membrane markers using cytofluorimetric analysis with a flow cytometer (Beckman Coulter, CA, USA; Yu et al., 2013 (link)). Cells were incubated for 20 min at 4°C with the following antibodies: anti-CD29, anti-CD90, anti-CD105 and anti-CD45 (Biolegend, CA, USA).

Flow cytometric analyses were performed with Gallios (Beckman Coulter) flow cytometers, and the data were analyzed with the FlowJo (Treestar) software packages.
MSCs were incubated with anti-CD90, anti-CD105, anti-CD73, anti-CD44, anti-CD11B, anti-CD34, anti-CD19, anti-CD45 and anti-HLA-DR antibodies to examine MSC surface markers, which were purchased from BioLegend. Anti-CD3, anti-CD4, anti-CD8, anti-TNF-α and anti-IFN-γ antibodies were used to examine stain T cells. Propidiumiodide (PI; BD) and Annexin V (BD) were used to stain apoptosis cells. Anti-CD86, anti-F4/80 and anti-CD45 antibodies were used to stain macrophages.

Isolation and identification of rat TSCs were performed as previously described [16 (link)]. Briefly, 0.3% pentobarbital sodium (Sigma, 30 mg/kg) was used for intraperitoneal anesthesia in rats. In sterile conditions, the tendon tissues were then removed, carefully dissected, cut into pieces, and digested in 3 mg/mL of type I collagenase (Sigma-Aldrich, St. Louis, MO, USA). After a 70-μm cell filter filtration, the suspension turned into a single cell suspension, which was then cultured in Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen, NY, Invitrogen Corporation, Grand Island, USA) containing 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin-streptomycin antibiotic mixture (Beyotime, Shanghai, China). Cells were subcultured at 80% confluence. Cells at passages three were incubated with fluorescein isothiocyanate-conjugated antibodies (anti-CD90, anti-CD105, anti-CD44, anti-CD11b, and anti-CD106) (Biolegend San Diego, CA, USA) through flow cytometry. The multilineage differentiation potential of TSCs was determined by inducing the differentiation of cells in passage 3 into osteocytes, adipocytes, and chondrocytes (all the differentiation media were from Cyagen).

The identification of ADSCs was tested by flow cytometry analysis. Briefly, three passaged ADSCs were collected and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD44, anti-CD90, phycoerythrin (PE)-conjugated anti-CD29, Alexa Fluor 647-conjugated anti-CD45, and Cyanine 7- conjugated anti-CD31 and their isotypic controls (all these antibodies were purchased from BioLegend, lnc.) respectively at room temperature for 30 min in dark. These cells were washed 3 times with PBS and tested by flow cytometry (Cytomics FC500 MPL, USA) and the results were analyzed with the FlowJo software. For neural differentiation, 70–80% confluent ADSCs were cultured in 10% FBS-containing DMEM/F12 supplemented with 5 µg/mL insulin, 200 µM indomethacin (INDO), and 500 µM IBMX for 48 h. The morphology changes were observed under an optical microscope and the expression of neural markers (NSE, GAP43, and S100β) was tested by Western Blotting.

Cells at passage three were incubated with fluorescein isothiocyanate-conjugated antibodies against surface markers, including anti-CD34 (Abcam, Cambridge, UK), anti-CD73 (BioLegend, San Diego, CA, USA), and anti-CD90 (BioLegend). Fluorescence was detected using a flow cytometer (NovoCyte 1300; ACEA, San Diego, CA, USA) to identify TSCs.
To detect collagen type I, TSCs were fixed with 4% paraformaldehyde (catalog number: P0099, Beyotime Biotechnology) and blocked with 20% heat-inactivated horse serum (Gibco) supplemented with 0.1% Triton-X 100 (Sigma-Aldrich). The cells were incubated with an anti-collagen type I antibody (catalog number: 14695-1-AP; Proteintech, Wuhan, China), followed by fluorescein-conjugated goat anti-rabbit IgG (catalog number: SA00003-11, Proteintech). The cells were then stained with 4′,6-diamidino-2-phenylindole (catalog number: P0131, Beyotime Biotechnology). Fluorescence was visualized under a fluorescence microscope (Leica, Wetzlar, Germany).