All tissue culture reagents and buffers were purchased from Gibco Laboratories, USA. Innohep was purchased from LEO Pharma, Denmark. All the fluorophore-conjugated monoclonal antibodies were purchased from BD Biosciences, USA, except for CD106 which was purchased from Invitrogen, Canada.
Cd106
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD106 is a laboratory instrument designed for the analysis and detection of cell surface markers. It utilizes flow cytometry technology to measure the expression of specific proteins, known as cluster of differentiation (CD) molecules, on the surface of cells. The CD106 provides researchers with a reliable and efficient tool for cellular analysis and characterization, enabling them to investigate various cellular properties and functions.
Automatically generated - may contain errors
Lab products found in correlation
Hla dr, by Thermo Fisher Scientific (3 mentions)
Cd105, by Thermo Fisher Scientific (2 mentions)
Fluorophore conjugated monoclonal antibodies, by BD (1 mentions)
Innohep, by Leo pharma (1 mentions)
Fcr blocker, by Miltenyi Biotec (1 mentions)
Cd144, by R&D Systems (1 mentions)
Cd309, by R&D Systems (1 mentions)
Cd271, by R&D Systems (1 mentions)
Fgfr 3, by R&D Systems (1 mentions)
Facsdiva 7, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Cd90f, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Alizarin red, by Cyagen (1 mentions)
Oil red o, by Cyagen (1 mentions)
Hla dr, by BD (1 mentions)
Cd140a, by R&D Systems (1 mentions)
Cd144, by Thermo Fisher Scientific (1 mentions)
Cd146, by Thermo Fisher Scientific (1 mentions)
Summit3, by Beckman Coulter (1 mentions)
10 protocols using cd106
1
Tissue Culture Immunophenotyping Protocol
2
Endothelial Marker Expression Analysis
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Receptor expression of typical endothelial markers was measured by flow cytometry at passage 3–5 for three matched donors. Cells were incubated with antibodies for 30 min at 4°C, washed with PBS supplemented with 0.1% bovine serum albumin and 0.1% sodium azide and then resuspended in the same buffer for flow cytometric analysis. The fluorescence was measured on a BD FACS Calibur and the results analyzed by Cell Quest software (Becton Immunocytometry Systems, Mountain View, CA, USA). PE-labelled antibodies and corresponding isotypes (mouse anti human) were used from BD Pharming unless otherwise stated: CD31 (WM59, IgG1), CD34 (581, IgG1), CD54 (HA58, IgG1), CD105 (SN6, IgG1, Invitrogen), CD106 (51-10C9, IgG1), CD144 (55-7H1, IgG1), CD309 (89106, IgG1), CD181 (5A12, IgG2b), CD182 (6C6, IgG1), CD183 (1C6, IgG1), CD184 (12G5, IgG2a, R&D systems), CD192 (48607, IgG2b, R&D systems), IgG1 (MOPC-21), IgG2a (G155-178), IgG2b (27–35), FcR Blocker (Miltenyi Biotec).
3
Immunophenotyping of UC-derived MSCs
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The immunoprofile of the UC cell populations at P3 (n = 3) was assessed using flow cytometry, as previously described [8 ]. Unless otherwise stated all antibodies were purchased from Becton Dickinson & company (BD), Oxford, UK. In brief, cells were stained with the following antibodies against CD14, CD19, CD34, CD45, HLA-DR, CD73, CD90, CD105 [16 (link),17 (link)]. Cells were also assessed for other markers indicative of MSC-related behaviour (CD271, Receptor Tyrosine Kinase-like Orphan Receptor 2 (ROR2) and Fibroblastic Growth Factor Receptor 3 (FGFR3), all R&D systems, Abingdon, UK), putative chondrogenic markers (CD151, CD39, CD44, CD49c, CD163, CD166) and immunomodulatory markers, CD106 and CD317 (eBioscience UK) [[18] (link), [19] (link), [20] (link)]. Co-stimulatory markers CD40, CD80, CD86 were also assessed, before and after stimulation with inflammatory cytokines, as well as HLA-DR, CD39, CD73, CD106 and CD317 as these markers are known to change upon inflammatory stimulus [21 (link)]. Appropriate isotype-matched IgG controls were used throughout, analysing approximately 100,000 cells for each antibody using a FACSCanto II flow cytometer and FACS DIVA 7 software (BD).
4
Isolation and Characterization of hUCMSCs
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Institutional Review Board approval was obtained for all the procedures. Following the written informed consent of parents, fresh human umbilical cords were obtained after birth from the Second Affiliated Hospital of Jinan University. hUCMSCs were isolated and cultured as previously described (16 (link)). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS), 100 U/ml penicillin G and 100 mg/ml streptomycin and maintained for 3–5 days in a humidified incubator at 37°C with 5% CO2. Flow cytometry on an Altra (Beckman Coulter, Fullerton, CA, USA) was performed to analyze characteristic MSC markers including CD29, CD34, CD90F, CD45, CD106 and HLA-DR (eBioscience, Inc., San Diego, CA, USA). hUCMSCs were also characterized by differentiation toward adipogenic and osteogenic lineages using previously described protocols (Alizarin red and Oil Red O assay) by commercial kits (Cyagen Biosciences Inc., Santa Clara, CA, USA).
5
Multimarker Immunophenotyping of SVF Cells
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Cells from the SVF at passage 3 were immunophenotyped by flow cytometry using cell surface markers CD14, CD34, CD45, CD73, HLA-DR (BD Pharmigen, EEUU), CD29, CD31, CD44, CD49D, CD19, CD90, CD105, CD106, CD133, CD144, CD146, (eBioscience), CD140A, CD140B, CD166 (RD Systems, EEUU). The clone, fluorochrome and amount of each antibody are provided in S1 Table . Briefly, 106cells/ml were resuspended in blocking buffer solution containing PBS supplemented with 3.0% BSA, and incubated on ice for 10 minutes. Then, they were gated according to their granularity (SS) and size (FS). 1x105 cells were stained using the antibodies (0.25 μg x 106 cells in 100 μl PBS) against the above surface markers or the isotype-matched control antibody. Samples were analyzed on a FACScan CyAnTM High-speed ADP Analyzer (Beckman Coulter, CA. EEUU). Data acquisition and analysis were performed using SUMMIT 3 software (Beckman Coulter). Unstained cells were used to establish flow cytometer settings. Debris and cells/particles with auto-fluorescence were removed by using a threshold on the forward scatter. The original (RAW) set of data is shown in S1 Fig .
6
FACS Characterization of ADSCs
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ADSC were grown until confluent, trypsinized and pelleted by centrifugation at 200 g for five minutes. For fluorescence-activated cell sorting (FACS) analysis, approximately 2.5 × 105 cells were resuspended in 100 μl FACS buffer containing 10% FBS in PBS. For FACS analysis of surface markers, each sample was incubated for 30 minutes at 4°C with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies against the following surface markers: CD45, CD34, CD11b, CD31, CD19, CD90, CD44, CD71, CD29, CD73, STRO-1, HLA-ABC, HLA-DR, CD117, CD105, CD106 (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. After incubation, the labeled cells were diluted with 2 ml of FACS buffer, pelleted and resuspended in 500 μl of FACS buffer. Generally, approximately 104 cells were analyzed per sample using the BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Results were analyzed using FlowJo software.
7
Immunophenotyping of Cultured Cells
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The following monoclonal antibodies were employed for immune-phenotyping studies: CD105, CD90, and CD166 (R&D Systems, Minneapolis, MN), CD54, CD45, CD106, HLA-DR (eBioscience, San Diego, CA), and CD34 (Dako, Glostrup, Denmark). Isotype control antibodies were purchased from eBioscience (San Diego, CA). 105-106 cells were employed for each experiment. First, cells were detached by trypsin and 3% rat serum was used for blocking non-specific binding sites before treatment with specific monoclonal and isotype control antibodies in 100 µL PBS-BSA 3% for 1 hour at 4°C in dark. The cells were then fixed by 1% paraformaldehyde and analyzed by using flow cytometry (Partec, Münster, Germany). Gating was performed for forward cells and non-specific attachments were put away based on isotype control antibodies.12 (link)
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The following monoclonal antibodies were employed for immune-phenotyping studies: CD105, CD90, and CD166 (R&D Systems, Minneapolis, MN), CD54, CD45, CD106, HLA-DR (eBioscience, San Diego, CA), and CD34 (Dako, Glostrup, Denmark). Isotype control antibodies were purchased from eBioscience (San Diego, CA). 105-106 cells were employed for each experiment. First, cells were detached by trypsin and 3% rat serum was used for blocking non-specific binding sites before treatment with specific monoclonal and isotype control antibodies in 100 µL PBS-BSA 3% for 1 hour at 4°C in dark. The cells were then fixed by 1% paraformaldehyde and analyzed by using flow cytometry (Partec, Münster, Germany). Gating was performed for forward cells and non-specific attachments were put away based on isotype control antibodies.12 (link)
8
Flow Cytometry of Isolated BMSCs
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For flow cytometry of the isolated BMSCs, BMSC cell markers, including CD29, CD34, CD44, CD45, CD71, CD90 and CD106 (BD Biosciences, Franklin Lakes, NJ, USA) were considered positive, whereas CD45 was considered negative. The following mouse monoclonal CD antibodies were conjugated with fluorescein isothiocyanate (FITC; BD Biosciences): Anti-CD29 (eBioscience, San Diego, CA, USA; cat. no. 12–0291); -CD34 (eBioscience; cat. no. 12–0341); -CD44 (eBioscience; cat. no. 12–0441) -CD45 (eBioscience; cat. no. 11–0451), -CD71 (eBioscience; cat. no. 11–0711), -CD90 (eBioscience; cat. no. 11–0900); and -CD106 (eBioscience; cat. no. 11–4321). The surfaces of the fourth passage BMSCs were stained; BMSC suspensions (1×105) were mixed with 10 µl FITC, and incubated in a dark room at 4°C for 30 min. The cells were then analyzed using a flow cytometer (BD FACSDiva software version 6.1.3; BD ARIA II and Diva software; BD Biosciences).
9
Immunohistochemical Analysis of Embryonic Tissues
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Samples were excised away from the embryos before being were fixed in 10% formalin for 24 h, washed with 70% ethanol, and embedded in paraffin. 4 μm sections were cut and stained for HE. Immunohistochemical analysis was performed by using reagents specific for CK18 (Clone B23.1, prediluted, Ventana), CK22 (Clone MM-1012-02, prediluted, Immuno Bio), Calponin (Clone 760-4376, 1:2000, Ventana), CD34 (Clone QBEnd/10, prediluted, Ventana), Ki-67 (Clone IR626, prediluted, Dako), ERα (Clone SP1, prediluted, Ventana), PR (Clone 1E2, prediluted, Ventana), Her2 (Clone 4B5, prediluted, Ventana), p63 (Clone 4A4, prediluted, Ventana), human mitochondria (MAB1273, 1:200, Millipore), CD106 (MA5-16429, 1:100, Thermo Fisher) on the BenchMark ULTRA IHC/ISH System. For all immunohistochemical stainings, DAB was used as a chromogen.
10
Endothelial Cell Characterization via Flow Cytometry
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ECs were grown to confluence and exposed to exosomes and TNF-a, as described above. ECs were detached with Enzyme-Free PBS-based Cell Dissociation Buffer (Thermo Fisher Scientific) and labelled with fluorescein isothiocyanate (FITC)-conjugated mouse IgG1, IgG2b, mouse anti-human CD54 (Intercellular Adhesion Molecule 1, ICAM-1), and CD106 (Vascular Cell Adhesion Molecules 1, VCAM-1) (Thermo Fisher Scientific). Mean fluorescence intensity and percentage of positively stained cells was calculated using the Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific).
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