Cd31 fitc clone wm59

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CD31-FITC (clone WM59) is a fluorochrome-conjugated monoclonal antibody that binds to the CD31 antigen, also known as platelet endothelial cell adhesion molecule (PECAM-1). CD31 is expressed on the surface of endothelial cells, platelets, monocytes, and some T-cell subsets.

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CD31-FITC (69 citations) Clone WM59 (2 citations)

5 protocols using cd31 fitc clone wm59

Immunophenotyping of Multiple Myeloma Cells

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The immunophenotype of primary BM CD138⁺ cells, HMCLs and microenvironment cells was analyzed with the following mAbs:
CD38-APC (clone HIT2, code n. 560677, BD)
CD31-FITC (clone WM59, code n. 555445, BD)
CD39-APC (clone eBioA1, code n. 17-0399-41, eBioscence; San Diego, CA)
CD73-APC (clone CB73), produced in the Lab of one of the authors (FM) and FITC-conjugated by AcZon (Bologna, Italy)
CD203a-FITC (clone 3E8, kindly provided by J. Goding)
CD14-PE (clone M5E2, code n. 555398, BD).

Costa F., Toscani D., Chillemi A., Quarona V., Bolzoni M., Marchica V., Vescovini R., Mancini C., Martella E., Campanini N., Schifano C., Bonomini S., Accardi F., Horenstein A.L., Aversa F., Malavasi F, & Giuliani N. (2017). Expression of CD38 in myeloma bone niche: A rational basis for the use of anti-CD38 immunotherapy to inhibit osteoclast formation. Oncotarget, 8(34), 56598-56611.

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Characterization of Multipotent Stromal Cells

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The expanded MSC population at third passage in cell culture were characterized by morphology (spindle-shaped cells) and by immunophenotypic analysis for the expression of the following membrane markers: mouse anti-human CD14-PE (clone 61D3), CD29-FITC (clone TS2/16), CD90-FITC (clone eBIO5E10) and CD105-PE (clone SN6) (from eBioscience), CD31-FITC (clone WM59), CD34-FITC (clone 8G12), CD45-PerCP-Cy5.5 (clone MOPC-21) and CD146-PE (clone P1H12) (from BD Biosciences, San Jose, CA, USA). Detection of osteocalcin in MSC culture by flow cytometry was performed with mouse anti-human PE-conjugated monoclonal antibody (R&D system) or isotype control antibody according to the manufacturer’s protocol.

Daltro G.C., Fortuna V., de Souza E.S., Salles M.M., Carreira A.C., Meyer R., Freire S.M, & Borojevic R. (2015). Efficacy of autologous stem cell-based therapy for osteonecrosis of the femoral head in sickle cell disease: a five-year follow-up study. Stem Cell Research & Therapy, 6(1), 110.

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Characterization of MSC by Flow Cytometry

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The MSC population expanded in the third passage in cell culture was characterized by morphology (spindle-shaped cells) and by immunophenotypic analysis for the expression of the following membrane markers: mouse anti-human CD14-PE (clone 61D3), CD29-FITC (clone TS2/16), CD90-FITC (clone eBIO5E10), and CD105-PE (clone SN6) (from eBioscience), CD31-FITC (clone WM59), CD34-FITC (clone 8G12), CD45-PerCP-Cy5.5 (clone MOPC-21), and CD146-PE (clone P1H12) (from BD Biosciences, San Jose, CA, United States). The detection of osteocalcin in MSC cultures by flow cytometry was performed with mouse anti-human PE-conjugated monoclonal antibody (R&D system) or control isotype antibody according to the manufacturer’s protocol.

Teixeira T.R., Daltro G.D., Sberge F.L., Barreto E.S, & da Silva A.F. (2024). Therapy with bone marrow mesenchymal stem cells in bone regeneration in children with osteonecrosis secondary to sickle cell disease. Frontiers in Cell and Developmental Biology, 12, 1410861.

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Characterization of Endothelial Progenitor Cells

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At day 12 after differentiation, adherent cells were harvested using TrypleE and made into a single-cell suspension in EGM-2 medium. Cells were counted and aliquots of the cell suspension were prepared for antibody staining. FcR blocking reagent (Miltenyi Biotech) was added to prevent the non-specific binding of antibodies. Anti-human CD31 (CD31-FITC, clone WM59 from BD Pharmingen), CD144 (CD144-PE, clone 16B1 from eBioscience) and NRP-1 (NRP-1-APC, clone AD5-176 from Miltenyi Biotech) antibodies were used at concentrations that were titrated before use (Supplementary Table 2). Propidium iodide (PI, Sigma) was added to the cell suspension for dead cell staining. Flow cytometric detection of the cell surface antigens and cells sorting were performed on an LSR II and FACSAria (Becton Dickinson), respectively. Compensation was set by single-positive controls using cord blood–derived ECFCs. A gating of targeted cell population was determined based on fluorescent minus one (FMO) controls for each fluorescent color.

Prasain N., Lee M.R., Vemula S., Meador J.L., Yoshimoto M., Ferkowicz M.J., Fett A., Gupta M., Rapp B.M., Saadatzadeh M.R., Ginsberg M., Elemento O., Lee Y., Voytik-Harbin S.L., Chung H.M., Hong K.S., Reid E., O'Neill C.L., Medina R.J., Stitt A.W., Murphy M.P., Rafii S., Broxmeyer H.E, & Yoder M.C. (2014). Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony–forming cells. Nature biotechnology, 32(11), 1151-1157.

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Multicolor Flow Cytometry Analysis of MSC Markers

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Immunophenotype analysis of mesenchymal, endothelial, human leucocyte antigen (HLA) and hematopoietic markers was performed by multicolor flow cytometry on c-kit + -derived cells. After detachment using a non-enzymatic method, cells were resuspended in PBS containing 0.1% BSA (Gibco, USA) and 2 mM EDTA (Gibco, USA) and incubated in the dark for 15 minutes with suitable combinations of the following monoclonal antibodies or isotype-matched control monoclonal antibodies: c-kit-APC (clone YB5.B8), CD34-FITC (clone 581), CD45-PE (clone HI30), CD29-PE (clone MAR4), CD31-FITC (clone WM59), CD90-FITC (clone 5E10), CD130-PE (clone AM64), HLA-DR-FITC (clone G46-6), CD73-PE (clone AD2), (BD Pharmingen, Italy), CD146-FITC (clone 128018), CD200-FITC (clone 325516), KDR-PE (clone 89106), (R&D Systems, USA), Lineage-biotin (MiltenyiBiotec, Italy), HLA-G-PE (clone MEM-G/9), (Exbio Praha, Czech Republic), CD144-Alexa700 (clone 16B1, eBioscience, UK). Samples were then washed with 1 mL of washing buffer and centrifuged for 10 minutes at 400 x g at 4°C to remove unbound antibodies. Cells were resuspended in 250 µL of washing buffer and analyzed.

Gambini E., Perrucci G.L., Bassetti B., Spaltro G., Campostrini G., Lionetti M.C., Pilozzi A., Martinelli F., Farruggia A., DiFrancesco D., Barbuti A, & Pompilio G. (2018). Preferential myofibroblast differentiation of cardiac mesenchymal progenitor cells in the presence of atrial fibrillation. Translational research : the journal of laboratory and clinical medicine, 192.

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