Anti cd73 fitc

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Anti-CD73-FITC is a fluorochrome-conjugated antibody that binds to the cell surface protein CD73. CD73 is an enzyme involved in the hydrolysis of extracellular adenosine monophosphate (AMP) to adenosine. This antibody can be used for the identification and characterization of cells expressing CD73 in flow cytometry applications.

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Anti-CD73 (23 citations) CD73-FITC (18 citations) FITC anti-human CD73 (5 citations) FITC)-conjugated anti-human CD73 antibody (2 citations)

7 protocols using anti cd73 fitc

Immunophenotypic Analysis of T-MSCs

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To analyze cell phenotype, T-MSCs were washed with FACS buffer (0.5 FBS and 0.1% NaN₃ in PBS), blocked with 0.5 µg/ml purified rat anti-mouse CD16/CD32 (BD Pharmingen) at 4°C for 5 min and stained with FITC anti-CD11b (cat. no. ICRF44, mouse IgG₁, κ), FITC anti-CD45 (2D1, mouse IgG₁, κ), FITC anti-CD73 (AD2, mouse IgG₁, κ), FITC anti-CD90 (5E10, mouse IgG₁, κ), FITC anti-CD105 (43A3, mouse IgG₁, κ) and FITC mouse IgG₁, κ isotype (MOPC-21) antibodies (all BioLegend, Inc.) at 0.5 µg/ml for 20 min at room temperature. After staining, the cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich; Merck KGaA) in phosphate-buffered saline (PBS) to final 0.5% PFA. Stained cells were acquired using a Novocyte flow cytometer (ACEA Bioscience, Inc.). Acquired cells were analyzed by FlowJo software (v10, TreeStar, Inc.).

Choi D.W., Cho K.A., Kim J., Lee H.J., Kim Y.H., Park J.W, & Woo S.Y. (2021). Extracellular vesicles from tonsil-derived mesenchymal stromal cells show anti-tumor effect via miR-199a-3p. International Journal of Molecular Medicine, 48(6), 221.

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Evaluating Mesenchymal Stem Cell Markers

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The marker expression of BM-hMSCs unstimulated or stimulated using 1.3 nM rhHGF or 32 nM aMD4dY-PA22 for 3 days were analyzed via flow cytometry. Harvested cells were blocked with PBS supplemented with Human TruStain FcX (Biolegend) for 10 min on ice. FITC-anti-human CD90 (Biolegend), PE anti-human CD105 (Biolegend), PE anti-human CD34 (Biolegend), FITC-anti-human CD45 (Biolegend), or FITC-anti-CD73 (Biolegend) diluted 10 times with a cell stain buffer (PBS supplemented with 1% FBS) was added to cells and incubated for 1 h on ice. For the isotype controls, 1:10 diluted PE mouse IgG1 k Isotype (Biolegend) and FITC mouse IgG1 k Isotype (Biolegend) were utilized. After washing two times with a cell stain buffer, cells were analyzed using Attune NxT flow cytometer (Thermo Fisher Scientific).

Ito K., Matsuda Y., Mine A., Shikida N., Takahashi K., Miyairi K., Shimbo K., Kikuchi Y, & Konishi A. (2022). Single-chain tandem macrocyclic peptides as a scaffold for growth factor and cytokine mimetics. Communications Biology, 5, 56.

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Characterization of Extracellular Vesicle Surface Markers

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The presence of CD39 and CD73 was confirmed by bead-assisted flow cytometry as previously described [18 (link)]. Briefly, 10 μg of TEX in 100 μl PBS were captured with biotin-labeled anti-CD63 mAb (1:50, Biolegend, San Diego, CA) for 2 h at RT. TEX were then incubated with 10 μL of ExoCap™ Streptavidin magnetic beads (MBL International, Woburn, MA) for 2 h at RT. The beads/anti-CD63Ab/TEX complexes were washed using a magnet and re-suspended in 200 μl of PBS. 100 μl of the sample was stained with an anti-CD39-FITC (1:25, 328206, Biolegend), anti-CD73-FITC (1:25, 344016, Biolegend) or with the appropriate isotype control for 1 h at RT. Analysis was performed with the Gallios flow cytometer and the Kaluza 1.0 software (Beckman Coulter, Krefeld, Germany). 10.000 events were acquired by gating on the bead fraction in the forward/sideward light scatter.

Ludwig N., Yerneni S.S., Azambuja J.H., Gillespie D.G., Menshikova E.V., Jackson E.K, & Whiteside T.L. (2020). Tumor-derived exosomes promote angiogenesis via adenosine A2B receptor signalling. Angiogenesis, 23(4), 599-610.

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Characterization of Adipose-Derived Stem Cells

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Flow cytometry was used to observe the ADSC phenotypes. Passage 3 ADSCs were trypsinized and incubated with anti-CD34-PE, anti-CD44-PE, anti-CD73-FITC, anti-CD90-FITC, and anti-HLA-DR-PE (Biolegend, San Diego, CA, USA). After three washes with PBS, the fluorescence of ADSCs was observed. To verify adipogenic differentiation and osteogenic differentiation, ADSCs at passage 3 were incubated separately with adipogenic and osteogenic differentiation medium (Gibco, United States) following the supplier’s instructions for 2 or 3 weeks. The induced cells were stained with Oil Red O and Alizarin Red respectively and microscopically imaged.

Zhou N., Xu Z., Li X., Ren S., Chen J., Xiong H., Wang C., Guo J., Kang Y., Chen Z., Li W., Yang X., Zhang X, & Xu X. (2022). Schwann Cell-Derived Exosomes Induce the Differentiation of Human Adipose-Derived Stem Cells Into Schwann Cells. Frontiers in Molecular Biosciences, 8, 835135.

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Isolation and Characterization of ADSCs

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Human adipose tissues and foreskins were obtained in accordance with procedures approved by the Ethics Committee at the Tongji Medical Collage of Huazhong University of Science and Technology. Primary fibroblasts were isolated from foreskins derived from routine pediatric circumcisions, using previously described protocols [26 (link)]. ADSCs were isolated and cultured in accordance with our established protocol [27 (link)]; ADSCs at passages 3–8 were used for experiments. Flow cytometry was conducted to identify ADSC phenotypes. ADSCs were incubated for 1 h with anti-CD34-BV421, anti-CD44-APC, anti-CD105-PE, anti-CD73-FITC, anti-CD31-FITC, and anti-CD90-FITC antibodies (all from Biolegend, San Diego, CA, USA); fluorescence was observed thereafter. For multi-lineage differentiation potential assays, ADSCs were incubated separately with adipogenic medium and osteogenic differentiation medium (Cyagen Biosciences Inc., Guangzhou, China). Cells were stained with Oil Red O and Alizarin Red, respectively. Subsequently, the stained cells were imaged using a microscope.

Ren S., Chen J., Guo J., Liu Y., Xiong H., Jing B., Yang X., Li G., Kang Y., Wang C., Xu X., Liu Z., Zhang M., Xiang K., Li C., Li Q., Machens H.G, & Chen Z. (2022). Exosomes from Adipose Stem Cells Promote Diabetic Wound Healing through the eHSP90/LRP1/AKT Axis. Cells, 11(20), 3229.

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Characterization of Human Bone Marrow-Derived Mesenchymal Stem Cells

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Healthy human bone marrow-derived mesenchymal stem cells (BM-MSCs, passage 3) were purchased (Gibco, #A15652) and cultured in low-glucose DMEM (Thermo Fisher Scientific, #11054020) supplemented with 10% Fetal Bovine Serum (FBS - Pan Biotech, #P30-3306), 1% L-glutamine (2 mM, Sigma Aldrich, #G7513) and penicillin/streptomycin antibiotic solution (100 U/0.1 mg/ml, Sigma Aldrich, #P4333) at 37 °C degrees containing 5% CO₂ in humid environment. For the evaluation of cell surface markers, cells were detached with trypsin-EDTA (Thermo Fisher Scientific, #25300054), collected by centrifuging at 300 X G for 5 minutes, and then suspended in Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% sodium azide. Next, the cells were labeled with anti-CD73 FITC (Clone:AD2, Biolegend, #344016), anti-CD90 PE (Clone: 5E10, Biolegend, #328110), anti-CD105 PerCP/Cy5.5 (Clone:43A3, Biolegend, #323216), anti-CD34 PE/Cy7 (Clone:581, Biolegend, #343515), anti-CD11b Alexa Fluor 700 (Clone:ICRF44, Biolegend, #301355), anti-HLA-DR Pacific Blue (Clone:L243, Biolegend, #307633), and anti-CD45 (Clone: J33, Beckman Coulter, #A96416) antibodies by incubating under dark conditions for 15 minutes at room temperature. The cells were immediately acquired on Beckman Coulter DxFLEX flow cytometry system. Analyses were performed on CytExpert software.

Aru B., Akdeniz T., Dağdeviren H., Gürel G, & Yanıkkaya Demirel G. (2023). Testosterone Propionate Promotes Proliferation and Viability of Bone Marrow Mesenchymal Stem Cells while Preserving Their Characteristics and Inducing Their Anti-Cancer Efficacy. Balkan Medical Journal, 40(2), 117-123.

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Immunophenotyping of Mesenchymal Stem Cells

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The expression of MSC surface markers were assessed by flow cytometry analysis. Cells were trypsinized into a single cell suspension and incubated with the fluorochrome-conjugated antibodies against human antigens, including anti-CD34-PE, anti-CD45-FITC, anti-CD90-FITC (BD Biosciences), anti-CD73-FITC (Bio Legend) and anti-CD105-FITC (R&D systems) for 15 mins in the dark at RT. After incubation, cells were washed with 1X PBS, centrifuged at 1500 rpm for 5 minutes and suspended in 1X PBS for flow cytometry analysis. Samples were acquired on a FACS canto II flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).

Kandoi S., L P.K., Patra B., Vidyasekar P., Sivanesan D., S. V., K. R, & Verma R.S. (2018). Evaluation of platelet lysate as a substitute for FBS in explant and enzymatic isolation methods of human umbilical cord MSCs. Scientific Reports, 8, 12439.

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