FACS experiments were performed with eBiosciences antibodies: anti-human Stro-1-FITC (340105), anti-human CD90-PE (12-0909-42), anti-human CD105-APC (17-1057-41), anti-mouse CD105-APC (17-1051-82), anti-human CD117- PE-Cy5 (15-1178-41), anti-human Ki67-PECy7 (25-5699-41), anti-human EpCAM-FITC (53-8326-41), and anti-human EpCAM-PE (12-9326-41); and BD antibody: anti-human Annexin V (BD 556422). EpCAM (130-061-101, Miltenyi Biotec) and CD45 (130-045-801, Miltenyi Biotec) beads were used to negatively select for epithelial and immune cells, respectively, prior to FACS. BD LSRII was used to collect the data for analysis using FlowJo software v10.3. EpCAM+ cells were gated for measuring epithelial CD105, AnnexinV, or Ki67 expression in 3D co-cultures.
Anti human cd90 pe
Manufactured by BD
Sourced in United States
Anti-human CD90-PE is a fluorescently-labeled monoclonal antibody that binds to the CD90 (Thy-1) antigen expressed on the surface of certain cell types. It is a research-use only product intended for flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti human stro 1 fitc, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd105 apc, by BD (2 mentions)
Anti human cd117 pe cy5, by BD (2 mentions)
Anti human epcam fitc, by BD (2 mentions)
Anti human epcam pe, by BD (2 mentions)
Epcam 130 061 101, by Miltenyi Biotec (2 mentions)
Cd45 130 045 801, by Miltenyi Biotec (2 mentions)
Software v10, by FlowJo (2 mentions)
Anti human cd105 apc, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Clone 5e10, by BD (1 mentions)
Goat anti mouse pe, by BD (1 mentions)
Clone wm59, by BD (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
4 6 diamidin 2 phenylindol dapi, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Anti human cd34 fitc, by BD (1 mentions)
Anti human cd31 fitc, by BD (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Anti cd105 pe, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti human cd90 pe
1
Comprehensive FACS Analysis of 3D Co-Cultures
2
Comprehensive FACS Analysis of 3D Co-Cultures
3
Characterizing primary hiPSCs and derivatives
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For cell characterization, 1 x 105 primary hiPSCs or hiPSC-derived cells were seeded in 12-well plates, grown to 50 - 70% confluence and fixed with 4% formaldehyde for 10 minutes at room temperature. Cells were washed in PBS and permeabilized in citrate buffer and blocked with 1x Dako wash buffer/10% FBS (S300685-2, Agilent). Anti-human CD90-PE (40 ng/µl, clone 5E10, BD Biosciences), anti-human CD31-PE (12.5 ng/µl, clone WM59, BD Biosciences), anti-human Cytokeratin 14 (4 ng/µl, clone LL001, Santa Cruz), anti-human Cytokeratin 5 (20 ng/µl, clone 2C2, Thermo Fischer) primary antibodies and appropriately titrated isotype controls were applied overnight at 4°C. As secondary antibody, a goat anti-mouse PE (40 ng/µl, BD Biosciences) was applied for 1 hour at room temperature. Cell nuclei were stained with 4′,6-Diamidin-2-phenylindol (DAPI, 1:1000, D1306, Molecular Probes) at room temperature for 10 minutes. In situ reporter staining during endothelial cell differentiation was performed by adding 125 ng/ml of anti-human CD31-PE (clone WM59, BD Biosciences) antibody in basal medium and incubating for 30 min at 37°C. After washing the cells with basal medium, EGM-2 / 10% hPL was added back before reporter staining was analyzed by fluorescence imaging.
4
Phenotypic Analysis of 3D Stem Cells
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The surface MSC phenotype was analyzed using flow cytometry analysis. 3D Petri dishes were inverted and centrifuged at 300 g for 10 min to remove spheroids from the agar. Spheroids and 2D MSCs were then Trypsinized using 2.5% Trypsin (Gibco) for 15 min, and 50,000 cells/tube were incubated with specific primary antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): FITC-anti-human CD31, FITC-anti-human CD34 (BD Bioscience, San Diego, CA, United States), PE-anti-human CD90, and PE-anti-human CD105 (BD Pharmingen, San Diego, CA, United States). Cells were also stained with the correlate IgG isotype controls conjugated with FITC (BD Bioscience) or PE (BD Pharmingen). Stainings were performed for 30 min at room temperature (RT) in the dark. Samples were run on a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, United States), and data were analyzed using FlowJo software (BD Biosciences).
5
Flow Cytometric Immunophenotyping of Cells
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After washing twice with PBS, cells were resuspended and incubated with pre–labelled antibodies for 15 min at room temperature. After two washes with PBS, cells were resuspended in 300 µL PBS and analyzed using a flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Histograms were generated using the cytexpert and FlowJo software. The antibodies used were as follows: FITC anti–human CD34 (Abcam, ab195013, Waltham, MA, USA), FITC anti–human CD45, (BD Biosciences, 557803, Piscataway, NJ, USA), PE–Cy7 anti–human CD14 (BD Biosciences, 561385, Piscataway, NJ, USA), PERCP–CY5.5 anti–human HLA–DR (BD Biosciences, 552764, Piscataway, NJ, USA), PERCP–CY5.5 anti–human CD73 (BD Biosciences, 561260, Piscataway, NJ, USA), PE anti–human CD90 (BD Biosciences, 555596, Piscataway, NJ, USA), APC anti–human CD19 (eBioscience, 11–0199042, San Diego, CA, USA), PE anti–CD105 (eBioscience, 25–1057–42, San Diego, CA, USA), PE anti–human CD44 (BD Biosciences, 555479, Piscataway, NJ, USA), APC anti–human CD29 (BD Biosciences, 559883, Piscataway, NJ, USA), and BV421 anti–human PDGFR–β (BD Biosciences, 564124, Piscataway, NJ, USA).
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