Cd90 pe

Manufactured by Bio-Rad

Sourced in United Kingdom

CD90-PE is a fluorochrome-conjugated antibody that specifically binds to the CD90 (Thy-1) cell surface antigen. CD90 is a glycophosphatidylinositol (GPI)-anchored protein expressed on various cell types, including hematopoietic stem cells, mesenchymal stem cells, and some subsets of T cells. The PE (phycoerythrin) fluorochrome allows for the detection and analysis of CD90-expressing cells using flow cytometry.

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6 protocols using cd90 pe

Immunophenotyping of Mesenchymal Stem Cells

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Immunophenotyping of MSC by flow cytometry was reported elsewhere[14 (link)]. Unless stated otherwise, fluorescence-conjugated monoclonal antibodies from Beckman Coulter were used. They were IgG1-FITC, IgG1-PE, HLA-DR-FITC, CD45-FITC, CD3-FITC, CD19-PE, CD16-FITC, CD33-FITC, CD38-FITC, CD34-PE, CD133-PE (Miltenyi Biotec GmbH, Germany), CD29-PE, CD44-FITC, CD73-FITC, CD90-PE, CD105-PE (Serotec, United Kingdom) and CD166-PE were used. At least 10000 events were acquired and signals were analysed by using the Coulter Epic XL MCL flow cytometer (Coulter, Miami, FL, United States).
Procedural details of immunofluorescence staining were described previously[15 (link)]. IgM anti-stage-specific embryonic antigen-4 (SSEA-4, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, United States), IgG_2b anti-octamer-binding transcription factor-4 (Oct-4; 1:100, Santa Cruz Biotechnology), IgG₁ anti-Nestin (1:400; BD Biosciences, San Francisco, CA, United States) were employed.
Cell viability was evaluated by using trypan blue dye exclusion test. Sterility check against microbial contamination was conducted at each MSC passage.

Tsang K.S., Ng C.P., Zhu X.L., Wong G.K., Lu G., Ahuja A.T., Wong K.S., Ng H.K, & Poon W.S. (2017). Phase I/II randomized controlled trial of autologous bone marrow-derived mesenchymal stem cell therapy for chronic stroke. World Journal of Stem Cells, 9(8), 133-143.

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Phenotypic profiling of MSCs and ECs

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Phenotypic characterisation was performed on culture expanded MSCs at different passages and on cultured ECs at p4 using: CD31-FITC (#MCA1738F), CD105-PE (#MCA1557PE), CD90-PE (#MCA90PE) (all from Serotec, Kidlington, UK), CD73-PE (#550257), CD146-PE (#550315) (both from BD Pharmingen, Oxford, UK), and CD271-PE (#130-091-885, Miltenyi Biotec). The isotype controls were IgG1-FITC (#550616, BD Pharmingen) and IgG1-PE (#MCA928PE, Serotec). A total of 2×10
⁵ cells was stained with 5 μl FITC- or PE-conjugated antibodies, and dead cells were excluded using 2 μg/ml propidium iodide (PI, #P1304MP, Invitrogen). Cells were acquired using FACScan equipped with CellQuest software version 3.1 (BD Biosciences) and the proportions of the different fractions were calculated as a percentage of total live cells.

Kouroupis D., Churchman S.M., McGonagle D, & Jones E.A. (2014). The assessment of CD146-based cell sorting and telomere length analysis for establishing the identity of mesenchymal stem cells in human umbilical cord. F1000Research, 3, 126.

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Flow Cytometry Analysis of hUC-MSC

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hUC-MSC-p3 and hUC-MSC-p15 were collected and treated with 0.25% trypsin. The cells were individually stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated anti-marker monoclonal antibodies in 100 μl PBS for 15 min at room temperature, or for 30 min at 4°C, as recommended by the manufacturer. The antibodies used were specific for the following human antigens: CD34-PE, CD44-FITC, CD45-PE, CD73-PE, CD90-PE and CD105-PE (10 μl for 1×10⁶ cells; AbD Serotec, Raleigh, NC, USA). Cells were analyzed on a Cytometer 1.0, Cytomics™ FC500 flow cytometry system (Beckman Coulter, Brea, CA, USA). Positive cells were counted and the signals for the corresponding immunoglobulin isotypes were compared (15 (link)).

ZHUANG Y., LI D., FU J., SHI Q., LU Y, & JU X. (2014). Comparison of biological properties of umbilical cord-derived mesenchymal stem cells from early and late passages: Immunomodulatory ability is enhanced in aged cells. Molecular Medicine Reports, 11(1), 166-174.

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Characterization of hBM-MSCs and hfC-MSCs

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hBM-MSCs or hfC-MSCs were stained with following anti-human monoclonal antibodies labelled with Fluorescence isothyocyanate (FITC) or phycoerythrin (PE): CD73-PE, CD90-PE, CD105-PE, CD117-PE, SSEA-4-PE, CD31-FITC, CD34-FITC, CD45-FITC, HLA-DR-FITC (all from Serotec), or isotype matched control monoclonal antibodies (Becton Dickinson). Stained cells were analyzed in Flow Cytometer (FACS Calibur, Becton Dickinson).

Garikipati V.N., Singh S.P., Mohanram Y., Gupta A.K., Kapoor D, & Nityanand S. (2018). Isolation and characterization of mesenchymal stem cells from human fetus heart. PLoS ONE, 13(2), e0192244.

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Flow Cytometry Phenotyping of Expanded Colonies

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Colonies expanded from blood were trypsinised, washed and stained with antibodies: CD31-FITC, CD90-PE, CD105-PE (AbD Serotec, Kidlington, UK), CD19-PE, CD33-FITC, CD34-PerCp, CD45-PE-Cy7, CD61-FITC, CD73-PE, (BD Biosciences, Oxford, UK) and CD271-APC (Miltenyi Biotec), at manufacturers recommended concentrations. Cells were washed, 30,000 events captured on a LSRII flow cytometer and the data analysed using FACSDiva Software (both BD Biosciences).

Churchman S.M., Jones E.A., Roshdy T., Cox G., Boxall S.A., McGonagle D, & Giannoudis P.V. (2020). Transient Existence of Circulating Mesenchymal Stem Cells in the Deep Veins in Humans Following Long Bone Intramedullary Reaming. Journal of Clinical Medicine, 9(4), 968.

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MSC Immunophenotyping by Flow Cytometry

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To prove cultures' MSC identity p1 and p3 cultures were trypsinised and stained with the following antibodies: CD90-PE, CD105-PE, CD31-FITC (all from Serotec, Kidlington, UK), CD73-PE, CD34-PerCp, CD45-PE-Cy7, CD19-FITC, CD14-PE, CD146-PE, and HLA-DR-FITC (all from BD Biosciences, Oxford, UK). All antibodies were used at manufacturers' recommended concentrations and the data were collected and analysed using a LSRII flow cytometer equipped with FACSDiva Software (both from BD Biosciences).

Churchman S.M., Boxall S.A., McGonagle D, & Jones E.A. (2017). Predicting the Remaining Lifespan and Cultivation-Related Loss of Osteogenic Capacity of Bone Marrow Multipotential Stromal Cells Applicable across a Broad Donor Age Range. Stem Cells International, 2017, 6129596.

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