Anti human cd73

Manufactured by BioLegend

Sourced in United States

Anti-human CD73 is a laboratory reagent used for the detection and analysis of CD73, a cell surface enzyme involved in the conversion of extracellular adenosine monophosphate (AMP) to adenosine. This product can be used in various immunological and cell biology applications, such as flow cytometry and immunohistochemistry, to identify and study cells expressing CD73.

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5 protocols using anti human cd73

Comprehensive Immune Profiling Protocol

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ViaStain AO/PI Staining Solution (catalog#CS2-0106) was purchased from Nexcelom. Propidium Iodide Solution (catalog#421301) was purchased from BioLegend. CellTrace CFSE Cell Proliferation Kit (catalog#C34554) was purchased from ThermoFisher. LIVE/DEAD Fixable Blue Dead Cell Stain Kit (catalog#L23105) was purchased from ThermoFisher. Anti-mouse CD73 antibody (clone: TY/23, catalog#BE0209), Anti-mouse B7H3 antibody (clone: MJ18, catalog#BE0124), anti-mouse PD-1 antibody (clone: RMP1-14, catalog#BE0146), anti-mouse CTLA-4 antibody (clone: UC10-4F10-11, catalog#BE0032), anti-mouse TIM-3 antibody (clone: RMT3-23, catalog#BE0115), anti-mouse TIGIT antibody (clone: 1G9, catalog#BE0274), anti-mouse LAG-3 antibody (clone: C9B7W, catalog#BE0174), anti-mouse GITR antibody (clone: DTA-1, catalog#BE0063), anti-mouse OX-40 antibody (clone: OX-86, catalog#BE0031), anti-mouse 401BB antibody (clone: LOB12.3, catalog#BE0169), anti-mouse NK1.1 antibody (clone: PK136, catalog#BE0036), anti-mouse CD4 antibody (clone: GK1.5, catalog#BE0003-1), and anti-mouse CD8 antibody (clone: 2.43, catalog#BE0061) were purchased from BioXcell. Anti-human PD-1 (Nivolumab, catalog#A2002) was purchased from Selleckchem. Anti-human CD73 (clone: AD2, catalog#344002) was purchased from BioLegend.

Xue G., Wang Z., Zheng N., Fang J., Mao C., Li X., Jin G., Ming X, & Lu Y. (2021). Elimination of acquired resistance to PD-1 blockade via the concurrent depletion of tumour cells and immunosuppressive cells. Nature biomedical engineering, 5(11), 1306-1319.

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Characterization and Multilineage Differentiation of UC-MSCs

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At P5, the UC-MSCs were characterized by flow cytometry using the following fluorescently labelled antibodies (BioLegend, USA): anti-human CD73 (cat. No. 344016), CD90 (cat. no. 328110), CD105 (cat. no. 323204), CD34 (cat. no. 343604), CD44 (cat. no. 103007), CD45 (cat. no. 304008), CD19 (cat. no. 302208), and HLA-DR (cat. no. 307606) antibodies as described previously.^{20 (link)} Cell phenotype was determined using a flow cytometer (Beckman Coulter, USA). For multipotent differentiation potential, human UC-MSCs were maintained in adipogenic (cat. no. A10070), chondrogenic (cat. no. A10071), and osteogenic (cat. no. A10072) differentiation media, respectively, following the manufacturer’s instruction (Gibco, USA). Successful inductions of adipogenesis, osteogenesis, and chondrogenesis were confirmed by staining with Oil Red O, Alizarin Red, and Alcian Blue (Gibco), and the expressions of specific differentiation genes were also detected by quantitative real-time polymerase chain reaction (qRT-PCR).

Zhang Q., Xiang E., Rao W., Zhang Y.Q., Xiao C.H., Li C.Y., Han B, & Wu D. (2021). Intra-articular injection of human umbilical cord mesenchymal stem cells ameliorates monosodium iodoacetate-induced osteoarthritis in rats by inhibiting cartilage degradation and inflammation. Bone & Joint Research, 10(3), 226-236.

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Immunophenotyping of Mesenchymal Stem Cells

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Cells were cultured in a 10-cm culture dish (Corning) at 70% confluency, and treated with accutase (Nacalai Tesque, Kyoto, Japan) to detach them from the culture dish. The cells were then fixed with 2% paraformaldehyde in PBS for 15 min, and blocked with 1% BSA (Wako) in PBS for 20 min. Subsequently, the cells were incubated (1 × 10⁶ cells per 100 μL) with PE-labeled anti-human CD90 (#BL328110, BioLegend, CA, USA), anti-human CD73 (#BL344004, BioLegend), anti-human CD34 (#BL343506, BioLegend), and mouse IgG1 kappa isotype control antibodies (#BL400114, BioLegend) for 1 h at room temperature in dark. Populations of 10,000 cells were analyzed using FACSVerse (BD Bioscience, CA, USA) and FACSuite software (BD Bioscience).

Nonaka K., Han X., Kato H., Sato H., Yamaza H., Hirofuji Y, & Masuda K. (2019). Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia. Biochemistry and Biophysics Reports, 19, 100648.

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Immunoregulatory Mechanism of CD4+ T cells

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The materials used were L-NMMA 1 mМ (Sigma), 1-МТ (Sigma), antihuman ICAM-1 SC-107L (Santa Cruz), antihuman IDO (H-110 sc-25808, Santa Cruz), antihuman iNOS (aa 781-798, Clone 2D2-B2, MAB9502, R&D Systems), antihuman CD28 NA/LE (BD 555725), antihuman CD3 NA/LE (BD 555336), antihuman CD4 (558116, BD PharMingen), antihuman CD25 (555346, BD PharMingen), antihuman CD45 (304026, BioLegend), antihuman CD3 (300431, BioLegend), antihuman CD90 (328110, BioLegend), antihuman CD 73 (344006, BioLegend), antihuman HLA-DR (307610, BioLegend), transwell permeable membranes 0.4 μm (Greiner 665640), TRIzol® Reagent (Invitrogen, 15596-018), CyQUANT®NF (C35006, Invitrogen), Maxima SYBR Green/ROX qPCR Master Mix (2X) (K0221 Thermo Scientific), SuperScript® III Reverse Transcriptase (Thermo Scientific), and SuperSignal™ West Pico Chemiluminescent Substrate (34078 Thermo Scientific).
All donors were notified and agreed to take part in the research according to the draft Declaration on Bioethics and Human Rights (SHS/EST/05/CONF.204/3 REV) paragraph 6 items b and c. All operational procedures and protocols were strictly followed in accordance with MSU bioethics committee guidelines. All experimental protocols were conducted according to the GLP rules signed by the Ministry of Health (Order number 267 from 19 June 2003).

Rubtsov Y., Goryunov К., Romanov А., Suzdaltseva Y., Sharonov G, & Tkachuk V. (2017). Molecular Mechanisms of Immunomodulation Properties of Mesenchymal Stromal Cells: A New Insight into the Role of ICAM-1. Stem Cells International, 2017, 6516854.

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Comprehensive Immune Profiling Protocol

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