Cells were analyzed by flow cytometry (Coulter FC500, Beckman Coulter, Brea CA) using the following antibodies: 1) human samples—CD34, CD19, HLA-DR, CD90 (all BD Biosciences, San Jose CA), CD44 (both Biolegend, San Diego CA), CD14, CD45 (AbB Serotec, Raleigh NC), CD105 (Abcam, Cambridge, MA); 2) porcine samples–CD44 (Biolegend), CD90 (antibodies-online.com Atlanta, GA), CD105 (Abcam), CD79a, CD45, SLA-DR (all AbD Serotec). Matched isotype controls were analyzed in parallel.
Cd105
Manufactured by Abcam
Sourced in United States, United Kingdom
CD105 is a transmembrane glycoprotein that functions as an endoglin, a component of the transforming growth factor-beta receptor complex. It plays a role in the regulation of cell proliferation, differentiation, and migration.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Cd11b, by Abcam (5 mentions)
Hla dr, by Abcam (3 mentions)
Cd146, by Abcam (3 mentions)
Cd166, by Abcam (3 mentions)
Cellquest software, by BD (2 mentions)
Cxcr4, by R&D Systems (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Huxmd 90011, by Cyagen (2 mentions)
Huxuc 90011, by Cyagen (2 mentions)
Huxma 90011, by Cyagen (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti cd9, by Abcam (2 mentions)
68 protocols using cd105
1
Flow Cytometry Immunophenotyping of Human and Porcine Cells
2
Immunofluorescence Staining of Stem Cells
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Cells were were fixed in 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.1% Triton X-100 for 30 min, and then incubated with blocking solution (PBS + 1% BSA) at 4°C for 30 min followed by primary antibodies at 4°C overnight. Primary antibodies were diluted in Immnol Fluorence Staining Primary Antibody Dilution Solution (Beyotime, P0108) at the followings ratios: OCT4 (rabbit polyclonal antibody, 1:500, Abcam), SOX2 (rabbit polyclonal antibody, 1:500, Abcam), NANOG (rabbit polyclonal antibody, 1:500, Abcam), SSEA4 (rabbit polyclonal antibody, 1:500, Abcam), CD105 (rabbit monoclonal antibody, 1:500, Abcam), β-Ⅲ-tubulin (rabbit monoclonal antibody, 1:500, Chemicon), AFP (mouse monoclonal antibody, 1:500, Chemicon), Desmin (mouse polyclonal antibody, 1:500, Abcam), Nestin (rabbit monoclonal antibody, 1:200, Chemicon), PDX1 (rabbit monoclonal antibody, 1:500, Chemicon) and α-actin (rabbit monoclonal antibody, 1:200, Sigma). After washing three times with PBS, cells were incubated with Alexa Fluor 488 (donkey anti-mouse) or Alexa Fluor 546 (goat anti-rabbit) conjugated secondary antibody (1:500; Chemicon) for 1h at room temperature in the dark. Finally, DNA was stained with Hoechst33342 (Beyotime, C1005) for 3 min. Negative controls were processed the same way, except that the primary antibodies were replaced with blocking buffer.
3
Quantifying c-kit+ Stem/Progenitor Cells in BM-MNCs
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To measure the number of c-kit-positive (c-kit+) stem/progenitor cells in the freshly isolated BM-MNCs, we labeled cells with a PE-conjugated anti-mouse c-kit antibody (eBioscience, CA USA) for 30 min. Respective isotype controls were used as a negative control. After washing, quantitative flow cytometry analysis was performed using a FACSCalibur instrument (Becton Dickinson, USA)24 (link)25 (link). The acquired data was analyzed using Cell Quest software (Becton Dickinson).
BM-MNCs were also stained with goat and rat monoclonal antibodies against CD90 (Abcam, UK), CD105 (Abcam), CXCR4 (R&D Systems, USA), followed by their respective FITC-conjugated secondary antibodies. The expressions of CD90, CD105, and CXCR4 in BM-MNCs were analyzed by flow cytometry as described above.
BM-MNCs were also stained with goat and rat monoclonal antibodies against CD90 (Abcam, UK), CD105 (Abcam), CXCR4 (R&D Systems, USA), followed by their respective FITC-conjugated secondary antibodies. The expressions of CD90, CD105, and CXCR4 in BM-MNCs were analyzed by flow cytometry as described above.
4
Flow Cytometric Characterization of Stem Cells
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Cell surface proteins were measured by flow cytometry using previously described methods [43 (link)]. The hBM-MSCs, hUC-MSCs, and 3 different hLD-SCs were sequentially incubated with primary antibodies in refrigerated blocking buffer for 1 h and secondary immunofluorescent antibodies. Antibodies against PE-labeled CD34 (BD Biosciences, Franklin Lakes, NJ, USA), FITC-labeled CD90 (Abcam, Cambridge, MA, USA), and CD105 (Abcam) were used while the CD34-positive cells were also parallelly examined (Supplementary Figure S1A ). Cells incubated with a blocking buffer without primary antibodies were used as a negative control. A total of 10,000 events were evaluated with the BD FACScanto II (Becton Dickinson and Company) and analyzed using the FlowJo software (ver 10.6.1; Treestar, Ashland, OR, USA).
5
Immunohistochemical Analysis of Angiogenic Markers
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Expression levels of CD105, CXCR4 and SDF-1α were detected by immunohistochemistry. Sections were dewaxed, endogenous peroxidase was neutralized with 3% H2O2 and antigen retrieval was conducted by incubating the sections in 0.01 M citrate buffer (pH 6.0) for 40 min at 92°C, and in normal goat serum for 20 min at 37°C. Sections were incubated with rabbit polyclonal CD105 (1:200; Abcam; cat. no. ab107595), rabbit polyclonal CXCR4 (1:200) and rabbit polyclonal SDF-1α (1:200) overnight at 4°C. The sections were washed 3 times in PBS and incubated with polyclonal HRP-conjugated goat anti-rabbit IgG (1:1,000) secondary antibody at 37°C. Sections were revealed by 3,3′-diaminobenzadine (DAB) (Haoran Biotechnology Co., Ltd.), counterstained with hematoxylin (Haoran Biotechnology Co., Ltd.), dehydrated and mounted with neutral balsam (Noble Ryder Beijing Science and Technology Co., Ltd., Beijing, China). The positive areas were quantified as the percentage of the total area using Image Pro Plus 5.0. A negative control was obtained using PBS rather than a primary antibody.
6
Immunocytochemical Validation of Isolated Endothelial Cells
7
Immunofluorescence Characterization of MSCs
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To analyze the MSCs biomarkers, the cells were first cultured on slides coated with 1% gelatin. The next day, the cells were fixed in 4% formaldehyde for 20 min at room temperature 27 °C. Fixed cells were washed twice with phosphate buffer saline (PBS) and blocked with 1% bovine serum albumin (Sigma) for 1 h at 27 °C. The cells were incubated with primary antibodies CD105 (Abcam, San Francisco, CA, USA), CD90 (Abcam), CD73 (Abcam), paxillin (Transduction Laboratories, San Jose, CA, USA), and focal adhesion kinase (FAK; Biolegend, San Diego, CA, USA) overnight at 4 °C. Secondary antibody (IgG(H+L)TRITC purchased from Jackson Immuno Research, West Grove, PA, USA was further incubated with the cells for 1 h at 27 °C. The cells on the slides were scanned using the TissueGnostics GmbH FACS‐like Tissue Cytometry (TissueFAXS Plus) imaging system (TissueGnostics, Vienna, Austria). Images were taken for each fluorescence channel with the field of view (FOV), and the percentage of positive cells were analyzed using the TissueQuest (analysis module for immunofluorescence staining) software based on the images taken.
8
Characterization of Mesenchymal Stem Cells
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Bone marrow mesenchymal stem cells (BMSCs, Institute of Hematology, Peking University People's Hospital, Beijing, China) and human placental mesenchymal stem cells (HPMSCs, Boya Stem Cell Bank, Beijing, China) were placed in a complete medium of DMEM/F-12 (GIBCO, USA) containing 10% of fetal bovine serum (Hyclone, USA) and 1% penicillin-streptomycin (GIBCO, USA), before being transferred to a cell culture incubator. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed once every two days (2 d). The cell growth status was observed under a microscope. When the confluence density of the cells reached 80–90%, they were passaged using routine subculture techniques, and some cells were cryopreserved at the same time. The cells were passaged continuously, and subsequent experiments were performed using P6–P9 generations of the cells. The content of MSC characterization included induction of adipogenic differentiation, induction of osteogenic differentiation, and the presence of surface markers of MSCs including CD105, CD73, CD34, CD11b, CD19, CD45, and HLA-DR (Abcam, USA) (Figure 1(a) ).
9
Characterization of pDGSCs by Flow Cytometry
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pDGSCs (P1) were trypsinized and counted with hemacytometer (Hausser Bright-Line, USA). Cells (5 × 105) for each antibody and negative control were taken into FACS tubes. They were incubated with the antibodies for CD34 (BD Pharmingen, Philippines), CD45 (BD Pharmingen, Philippines), CD105 (Abcam, USA), CD90 (BD Pharmingen, Philippines), and CD44 (Abcam, USA) cell surface markers at room temperature for 1 h. Cells were washed with physiological saline solution and centrifuged at 2,200 rpm for 5 min to remove excess antibodies. After centrifugation, they were resuspended in 400 μL physiological saline solution and samples were analyzed in flow cytometer (FACSCalibur, Becton Dickinson, USA).
10
Stem Cell Surface Marker Identification
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Cells at the 5th passage of culture in each oxygen tension were detached from flasks using Accutase (Sigma-Aldrich). For identification of stem cell surface markers, cells (1 × 105) were labeled with antibodies against the surface markers CD105 (FITC, mouse, Abcam, Cambridge, UK), CXCR4 (FITC, mouse, R&D systems, USA), G-CSFR (FITC, mouse, R&D systems, USA), IgG1 isotype control (FITC, mouse, Biolegend, USA) and IgG2a isotype control (FITC, mouse, Santa Cruz, USA) for 1 hour, at 4 °C. Labeled cells were analyzed using FACSAria II flow cytometer (Becton Dickinson, USA). Only viable cells as determined by propidium iodide (PI) (Sigma-Aldrich) exclusion were gated and analyzed.
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