Cryopreserved PBMC were isolated and used for immunophenotypic staining as previously described (108 (link)). Cells were stained with Zombie NIR fixable viability stain (BioLegend, San Diego, USA) and the following anti-human monoclonal fluorochrome-conjugated antibodies: anti-CD45RA, anti-CD4, anti-TCR-γ/δ (BD Biosciences, Heidelberg, Germany), anti-TCR-Vδ2 (Beckman Coulter Life Sciences, Indianapolis, USA), anti–HLA-DR, anti-CD27, anti-CD279 (PD-1), anti-TIGIT, anti-CD8, anti-CD28, anti-CD39, anti-CD38, anti-CD19, anti-CD3, anti-CD73 and anti-CD14 (all BioLegend) (Supplementary Table 2
Anti cd73
Manufactured by BioLegend
Sourced in United States
Anti-CD73 is a monoclonal antibody that binds to the CD73 (ecto-5'-nucleotidase) cell surface protein. CD73 is an enzyme that catalyzes the conversion of extracellular adenosine monophosphate (AMP) to adenosine, which has immunosuppressive effects.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd90, by BioLegend (14 mentions)
Anti cd105, by BioLegend (11 mentions)
Anti cd45, by BioLegend (11 mentions)
Anti cd34, by BioLegend (8 mentions)
Anti cd11b, by BioLegend (7 mentions)
Anti cd19, by BioLegend (6 mentions)
Anti hla dr, by BioLegend (5 mentions)
Anti cd44, by BioLegend (5 mentions)
Anti cd31, by BioLegend (5 mentions)
Anti cd4, by BD (4 mentions)
Anti cd39, by BioLegend (3 mentions)
Anti cd3, by BioLegend (3 mentions)
Anti cd8, by BioLegend (3 mentions)
Heracell 150i, by Thermo Fisher Scientific (3 mentions)
Anti tcrγδ, by BD (2 mentions)
Anti tcr vδ2, by Beckman Coulter (2 mentions)
Anti t bet, by BioLegend (2 mentions)
Anti foxp3, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Anti cd29, by BioLegend (2 mentions)
23 protocols using anti cd73
1
Immunophenotyping of Cryopreserved PBMCs
2
Comprehensive Immune Profiling of Tumor-Bearing Mice
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The following Abs were used: anti-CTLA-4 (HMCD15201, Thermo Fisher), anti-CD39 (143804, BioLegend), anti-CD8 (553031, BioLegend), anti-CD73 (127220, BioLegend), anti-Tbet (644810, BioLegend), anti-CD44 (103030, BioLegend), anti-KLRG1 (138414, BioLegend), anti-CD11b (101230, BioLegend), anti-CXCR5 (551961, BD Biosciences), anti-CD25 (564571, BD Biosciences), anti-CD4 (553052, BD Biosciences), anti-CD107a/b (553793/558758, BD Biosciences), anti-B220 (561102, BD Biosciences), anti-PD1 (11-9985-81, eBioscience), anti-Foxp3 (50-5773-82, eBioscience), anti-CD11c (17-0114-82, eBioscience), anti-CD45 (12-0451-82, eBioscience), anti-CD49b (25-5971-82, eBioscience) and anti-TCF-1 (2206S, Cell Signaling Technology). Fixable Viability Dye (65-0865-14, eBioscience)-stained cells were excluded from analysis. Tumor-bearing mice were sacrificed; the tumors were sliced and digested with type I collagenase (A004194-0001, Sangon Biotech) for 1 hour at 37°C; and the spleens and dLNs were ground to generate single-cell suspensions. The single-cell suspensions were filtered through 70 µm strainers (352350, BD Biosciences) and stained as described. The stained cells were evaluated by BD FACS Canto II flow cytometry, and the flow cytometry data were analyzed with FlowJo software (Tree Star).
3
Characterization of hucMSC Surface Markers and Differentiation
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To evaluate the surface markers of hucMSCs, hucMSCs at passage three were incubated with antibodies anti-CD34 (catalog number: 343503, BioLegend, San Diego, CA, USA), anti-CD73 (catalog number: 344015, BioLegend), anti-CD29 (catalog number: 303004, BioLegend), anti-CD14 (catalog number: 397706, BioLegend), anti-CD105 (catalog number: 323203, BioLegend), anti-CD19 (catalog number: 302205, BioLegend), anti-CD45 (catalog number: 304005, BioLegend), anti-HLA-DR (catalog number: 327005, BioLegend), and anti-CD90 (catalog number: 328108, BioLegend). Fluorescence was detected using a flow cytometer (NovoCyte 1300; ACEA, San Diego, CA, USA) to identify hucMSCs.
To evaluate cell differentiation, hucMSCs at passage three were cultured in adipogenic (catalog number: A1007001, Gibco, Grand Island, USA), chondrogenic (catalog number: A1007101, Gibco), or osteogenic medium (catalog number: A1007201, Gibco) for 3 weeks. Subsequently, the cells were fixed with 4% paraformaldehyde, and stained with oil red O (catalog number: HY-D1168, MedChemExpress, New Jersey, USA), alcian blue (catalog number: HY-D0001, MedChemExpres), or alizarin red (catalog number: HY-120601, MedChemExpres), respectively. The stained cells were observed under a light microscope (Olympus, Tokyo, Japan).
To evaluate cell differentiation, hucMSCs at passage three were cultured in adipogenic (catalog number: A1007001, Gibco, Grand Island, USA), chondrogenic (catalog number: A1007101, Gibco), or osteogenic medium (catalog number: A1007201, Gibco) for 3 weeks. Subsequently, the cells were fixed with 4% paraformaldehyde, and stained with oil red O (catalog number: HY-D1168, MedChemExpress, New Jersey, USA), alcian blue (catalog number: HY-D0001, MedChemExpres), or alizarin red (catalog number: HY-120601, MedChemExpres), respectively. The stained cells were observed under a light microscope (Olympus, Tokyo, Japan).
4
Characterization of Aged MSCs with MIF
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The surface antigen expression of MSCs was determined by flow cytometry. The following antibodies were used: anti-CD29 (Biolegend, 303003), anti-CD45 (Biolegend, 304011), anti-CD73 (Biolegend, 344003), anti-CD90 (Biolegend, 328107) and anti-CD105 (Biolegend, 323205). Aged MSCs were transfected with either a lentivirus containing MIF (MIF-aged MSCs) or a control vector (aged MSCs), as previously described [48 (link)]. The transfection efficiency was examined by fluorescence microscopy and Western blotting. The capacity of MSCs to differentiate into osteocytes and adipocytes was examined as previously reported [48 (link)].
5
Characterization of GMP-Grade Adipose MSCs
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Good Manufacturing Practices (GMP)-Grade human adipose-derived MSCs (Steminent Biotherapeutics Inc., Taiwan) were cultured in MSC maintenance medium consisting of IMDM, 10% FBS (#10270106, Gibco®, Thermo Fisher Scientific, Waltham, MA, USA), 10 ng/mL bFGF (#233-FB, R&D Systems, Minneapolis, MN, USA) and 1% PSG (#10378016, Gibco®). Human skin fibroblasts (#GM08429, Coriell Institute, Camden, NJ, USA) were cultured in alpha MEM supplemented with 15% FBS and 1% PSG at a seeding density of 3000 cells/cm2, and the cells were subcultured after reaching confluence. MSC characteristics were confirmed according to the minimal criteria defined by the International Society for Cell and Gene Therapy (ISCT) [35] (link). Surface markers were analyzed using a FACSAria Fusion Cell sorter and Cell Analyzer (BD Biosciences, San Jose, CA, USA). Primary antibodies for flow cytometry were as follows: anti-CD105, anti-CD90, anti-CD73, anti-CD34, anti-CD45 and anti-CD11b (#800505, #328107, #344015, #343607, #368511, #301309, BioLegend, San Diego, CA, USA; 1:100 dilution). Multidifferentiation capacities were assessed by alkaline phosphatase and Oil Red O, as previously described [9] (link), [36] (link).
6
Immunophenotyping of Mesenchymal and Immune Cells
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Flow cytometric analyses were performed with Gallios (Beckman Coulter) flow cytometers, and the data were analyzed with the FlowJo (Treestar) software packages.
MSCs were incubated with anti-CD90, anti-CD105, anti-CD73, anti-CD44, anti-CD11B, anti-CD34, anti-CD19, anti-CD45 and anti-HLA-DR antibodies to examine MSC surface markers, which were purchased from BioLegend. Anti-CD3, anti-CD4, anti-CD8, anti-TNF-α and anti-IFN-γ antibodies were used to examine stain T cells. Propidiumiodide (PI; BD) and Annexin V (BD) were used to stain apoptosis cells. Anti-CD86, anti-F4/80 and anti-CD45 antibodies were used to stain macrophages.
MSCs were incubated with anti-CD90, anti-CD105, anti-CD73, anti-CD44, anti-CD11B, anti-CD34, anti-CD19, anti-CD45 and anti-HLA-DR antibodies to examine MSC surface markers, which were purchased from BioLegend. Anti-CD3, anti-CD4, anti-CD8, anti-TNF-α and anti-IFN-γ antibodies were used to examine stain T cells. Propidiumiodide (PI; BD) and Annexin V (BD) were used to stain apoptosis cells. Anti-CD86, anti-F4/80 and anti-CD45 antibodies were used to stain macrophages.
7
Characterization of Human Umbilical Cord Blood-Derived MSCs
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MSCs were isolated from human UCB as previously described [17 (link)]. The protocol has been approved by Institutional Committee for Stem Cell Research (IC-SCR; Code: 2019-03-IMP-08). MSCs propagated to the 3rd-8th passages were characterized using accepted MSC-positive markers anti-CD73, CD90, and CD105 (BioLegend, San Diego, CA, USA), negative hematopoietic markers anti-CD45 and CD34 (BioLegend, San Diego, CA, USA), HLA-DR (BD Biosciences, San Jose, CA, USA) by confocal microscopy (FluoView F10i confocal microscope, Olympus), and flow cytometry (Becton Dickinson, New Jersey, USA). Negative gates were set relative to isotype controls. No chromosomal aberrations were observed at high passage (data not shown). In vitro differentiation was performed using StemPro™ Adipogenesis Differentiation Kit (A1007001, Thermo Scientific, Pittsburg, PA Scientific, USA), StemPro™ Osteogenesis Differentiation Kit (A1033201, Thermo Scientific, Pittsburg, PA Scientific, USA), and StemPro™ Chondrogenesis Differentiation Kit (A1007101, Thermo Scientific, Pittsburg, PA Scientific, USA) [19 (link), 20 ].
8
Comprehensive Immunophenotyping of Cultured Cells
9
Characterization of Tissue-Specific Stem Cells
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Cells at passage three were incubated with fluorescein isothiocyanate-conjugated antibodies against surface markers, including anti-CD34 (Abcam, Cambridge, UK), anti-CD73 (BioLegend, San Diego, CA, USA), and anti-CD90 (BioLegend). Fluorescence was detected using a flow cytometer (NovoCyte 1300; ACEA, San Diego, CA, USA) to identify TSCs.
To detect collagen type I, TSCs were fixed with 4% paraformaldehyde (catalog number: P0099, Beyotime Biotechnology) and blocked with 20% heat-inactivated horse serum (Gibco) supplemented with 0.1% Triton-X 100 (Sigma-Aldrich). The cells were incubated with an anti-collagen type I antibody (catalog number: 14695-1-AP; Proteintech, Wuhan, China), followed by fluorescein-conjugated goat anti-rabbit IgG (catalog number: SA00003-11, Proteintech). The cells were then stained with 4′,6-diamidino-2-phenylindole (catalog number: P0131, Beyotime Biotechnology). Fluorescence was visualized under a fluorescence microscope (Leica, Wetzlar, Germany).
To detect collagen type I, TSCs were fixed with 4% paraformaldehyde (catalog number: P0099, Beyotime Biotechnology) and blocked with 20% heat-inactivated horse serum (Gibco) supplemented with 0.1% Triton-X 100 (Sigma-Aldrich). The cells were incubated with an anti-collagen type I antibody (catalog number: 14695-1-AP; Proteintech, Wuhan, China), followed by fluorescein-conjugated goat anti-rabbit IgG (catalog number: SA00003-11, Proteintech). The cells were then stained with 4′,6-diamidino-2-phenylindole (catalog number: P0131, Beyotime Biotechnology). Fluorescence was visualized under a fluorescence microscope (Leica, Wetzlar, Germany).
10
Murine Immune Cell Profiling
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