Dnam 1

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Sourced in United States

DNAM-1 is a cell surface receptor molecule involved in the regulation of cell-cell adhesion and immune response. It functions as a co-stimulatory molecule that enhances T cell activation and proliferation. DNAM-1 plays a role in natural killer cell-mediated cytotoxicity and tumor surveillance.

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Lab products found in correlation

Nkp30, by BioLegend (3 mentions) Facs cant 2, by BD (2 mentions) Ctla 4, by BioLegend (2 mentions) Anti cd3, by BioLegend (2 mentions) Tim 3, by BioLegend (2 mentions) Facscalibur, by BD (1 mentions) Ifn γ, by BioLegend (1 mentions) Tnfα mab11, by Thermo Fisher Scientific (1 mentions) Cd62l dreg 56, by Thermo Fisher Scientific (1 mentions) Ulbp 1 170818, by R&D Systems (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Aldehyde sulphate latex beads, by Thermo Fisher Scientific (1 mentions) Cd112, by BioLegend (1 mentions) Nkp30, by Beckman Coulter (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions) Nkp44, by Miltenyi Biotec (1 mentions) Cytoflex s, by Beckman Coulter (1 mentions) Cx3cr1, by BioLegend (1 mentions) Tgfβrii, by R&D Systems (1 mentions) Goat anti mouse igg, by BioLegend (1 mentions)

8 protocols using dnam 1

Flow Cytometry Characterization of NK Cells

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For flow cytometry, single-cell suspensions were labelled with fluorescently conjugated anti-mouse antibodies. All antibodies were titrated and used at optimal dilutions. CD3 (145.2C11), CD27 (29A1.4), CD11b (M1/70.15), CD69 (H1.2F3), IFN-γ (XMG1.2), CD244 (m2B4(B6)458.1), cKit (2B8), CD127 (A7R34), CD122 (TM-β1), CD135 (A2F10), NK1.1 (PK136), KLRG1 (2F1), CD107a (1D4B), NKp46 (29A1.4), DNAM-1 (10E5, 480.1, TX-42.1 and 3B3 (ref. 28 (link))) and Ly49A (YEI/48) (Biolegend), Ly49G2 (4D11), and NKG2D (CX5) (BD Biosciences), and Ly49I (YLI-90) and NKG2A (20d5) (eBioscience). The 4LO3311 (Ly49C) hybridoma was a kind gift from Suzanne Lemieux. All surface staining was performed in phosphate-buffered saline (PBS) after blocking unspecific staining via FcγRII/III with purified anti-CD16/32 (2.4G2) (MabTech). Dead cells were excluded from the gating with the Aqua dead cell dye (Life Technologies). Flow cytometric analyses were performed on an LSRII or FACSCalibur (Becton Dickinson) and data analysis was performed using the FlowJo software (Tree Star). For comparison of relative expression levels of specific NK-cell subsets, we have normalized the expression data (MFI) of all NK-cell subsets in every individual mouse to the mean of the MFI of the same NK subset in all K^b−/−D^b−/− mice in the same experiment.

Wagner A.K., Kadri N., Snäll J., Brodin P., Gilfillan S., Colonna M., Bernhardt G., Höglund P., Kärre K, & Chambers B.J. (2017). Expression of CD226 is associated to but not required for NK cell education. Nature Communications, 8, 15627.

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Isolation and Analysis of Splenocytes

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Splenocytes were obtained from a previously defined patient with WAS due to complete deletion of exons 1–12 of WASp52 (link) or from a normal person undergoing splenectomy due to trauma. Control PBMCs were isolated from a healthy volunteer. Blood and the tissue were collected under protocols including written informed consent and approved by the Institutional Review Boards of the Immune Disease Institute (formerly the Center for Blood Research; Boston, MA, USA). PBMCs and splenocytes were used from cryopreserved samples where indicated. For flow cytometry, single-cell suspensions were labeled with fluorescently conjugated anti–human antibodies: CD56 (318334, Biolegend), CD3 (317328, Biolegend), DNAM-1 (338304, Biolegend), CD69 (562617, BD Biosciences), Perforin (556577, BD Biosciences), CD107a (15–107942, eBioscience), Granzyme (GRB17, Invitrogen), and IFNγ (502520, Biolegend). Expression data in neuroblastoma diffuse large B cell lymphoma patients was acquired from the R2 database (R2: Genomics Analysis and Visualization Platform; http://r2.amc.nl).

Kritikou J.S., Dahlberg C.I., Baptista M.A., Wagner A.K., Banerjee P.P., Gwalani L.A., Poli C., Panda S.K., Kärre K., Kaech S.M., Wermeling F., Andersson J., Orange J.S., Brauner H, & Westerberg L.S. (2016). IL-2 in the tumor microenvironment is necessary for Wiskott-Aldrich syndrome protein deficient NK cells to respond to tumors in vivo. Scientific Reports, 6, 30636.

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Phenotyping of Expanded Natural Killer Cells

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The phenotype of eNK cells was analyzed weekly using flow cytometry. The following mouse anti-human antibodies were used for staining: CD16 (3G8), CD56 (NCAM 16.2), NKG2D (1D11), DNAM-1 (DX11), CD3 (HIT3a) from BD PharMingen; CXCR1 (8F1), CXCR3 (CEW33D), CXCR4 (L276F12), CXCR6 (K041E5), CCR4 (L291H4), CCR5 (J418F1), CCR6 (G034E3), CCR9 (L053E8), CX3CR1 (2A9-1), Fas-L (NOK-1), IFNγ (B27) from BioLegend; CD62L (DREG-56), intracellular Perforin (dG9), Granzyme B (GB11), and TNF-α (MAb11) from eBioscience. For surface staining, NK cells were resuspended in FACS buffer (0.5% FBS and 2 mM EDTA in PBS), blocked with 10% mouse serum for 30 min, then incubated with the indicated antibodies for 30 min at 4°C using concentrations recommended by the manufacturers. For subsequent intracellular staining, NK cells were stained using the BD Cytofix/Cytoperm Kit following manufacturer protocol. Data was acquired using a BD FACS Accuri C6 and analyzed using FlowJo v10.2 software. Isotype IgG antibodies were used as negative controls. Human NK cells were defined as CD16⁺, CD56⁺ and CD3⁻. Mouse anti-human HLA-ABC (W6/32), MICA/B (6D4), CD155 (2H7) and PD-L1 (MIH1) from eBioscience, ULBP-1 (170818) from R&D, and Fas (DX2) from BioLegend were used to determine MHC-I, NKG2D ligand and DNAM-1 ligand expression on TC106 with surface staining performed as above for NK cells.

Tong A.A., Hashem H., Eid S., Allen F., Kingsley D, & Huang A.Y. (2017). Adoptive natural killer cell therapy is effective in reducing pulmonary metastasis of Ewing sarcoma. Oncoimmunology, 6(4), e1303586.

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Comprehensive lymphocyte profiling by flow cytometry

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Freshly isolated PBMCs were stained with antibodies specific to the cell surface markers of T cells, B cells, and NK lymphocyte, including anti-CD3, CD19, CD20, CD4, CD8, CD16, CD56, NKp46, NKG2D, NKp30, TLR4, DNAM-1, NKG2A, 4–1BB, OX40, ICOS, PD-1, CTLA4, GITR, LAG3, TIGIT, and TIM3 (BioLegend, San Diego, CA, USA). The stained cells were analyzed using a FACSCant II (BD Biosciences, San Jose, CA, USA), and the data were analyzed using the FlowJo software package (ver.10; Tree Star, Ashland, OR, USA).

Nakagami Y., Suzuki S., Espinoza J.L., Vu Quang L., Enomoto M., Takasugi S., Nakamura A., Nakayama T., Tani H., Hanamura I, & Takami A. (2019). Immunomodulatory and Metabolic Changes after Gnetin-C Supplementation in Humans. Nutrients, 11(6), 1403.

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Immunophenotyping of PBMCs

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Isolated PBMCs were stained with antibodies specific to the cell surface markers of NK lymphocytes, T cells, and B cells, including anti-CD3, CD19, CD20, CD4, CD8, CD16, CD56, NKp46, NKG2D, NKp30, TLR4, DNAM-1, NKG2A, 4-1BB, OX40, ICOS, PD-1, CTLA4, GITR, LAG3, TIGIT, and TIM3 (BioLegend, San Diego, CA, United States). The stained cells were analyzed using FACSCant II (BD Biosciences, San Jose, CA, United States), and the data were analyzed using the FlowJo software package (ver. 10; Tree Star, Ashland, OR, United States). In the PBMC gating, CD3⁺CD56⁻cells were defined as T cells, CD3⁻CD56 ⁺ cells were defined as NK cells, CD19 + CD20 ⁺ cells were defined as B cells, and T cells were further divided into CD4⁺ T cells and CD8⁺ cells.

Ogawa-Ochiai K., Ishikawa H., Li H., Vu Quang L., Kimoto I., Takamura M., Hongawa T., Hane Y., Suzuki S., Okajima M., Mori K., Ito M, & Takami A. (2021). Immunological and Preventive Effects of Hochuekkito and Kakkonto Against Coronavirus Disease in Healthcare Workers: A Retrospective Observational Study. Frontiers in Pharmacology, 12, 766402.

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Comprehensive NK Cell Phenotyping by Flow Cytometry

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NK cells were stained with antibodies against surface molecules for 30 min a 4 °C, washed and then analyzed by LX-Cytoflex or Cytoflex S flow-cytometers (Beckman) and FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA) was used for data analysis. For exosome analysis, 5 µg of exosomes were coated to 5 µL of aldehyde/sulphate latex beads (Invitrogen, Carlsbad, CA, USA) and following 15 min incubation, exosome-coupled beads were diluted with 1 mL in PBS and incubated overnight at 4 °C. The day after, samples were washed twice and incubated with conjugated antibodies for 30 min at 4 °C. After 3 washings, they were analyzed by flow-cytometry. For detection of cytotoxic proteins or inner proteins inside exosomes (Granzyme A-B, Perforin, IFN-γ and PD-1), samples were fixed in 4% paraformaldehyde and permeabilized in Triton 0.1% before proceeding with incubation at 37 °C with conjugated antibodies.
Antibodies used were CD3, CD81, CD63, NKp44, CD69, PD1, and CD54 (Miltenyi, Bergish Galdbach, Germany), CD56, CD16, Granzyme A and B (Beckton Dickinson, Franklin Lakes, USA), NKp46, IFN-γ (Thermo Fischer, Waltham, USA), NKG2D, DNAM1, Perforin, CD115, CD112 (Biolegend, San Diego, CA, USA), NKp30 (Beckman Coulter, Brea, CA, USA), CD45, CD14, and CD19 (Beckman Coulter).

Di Pace A.L., Tumino N., Besi F., Alicata C., Conti L.A., Munari E., Maggi E., Vacca P, & Moretta L. (2020). Characterization of Human NK Cell-Derived Exosomes: Role of DNAM1 Receptor in Exosome-Mediated Cytotoxicity against Tumor. Cancers, 12(3), 661.

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Phenotypic Characterization of TGF-β DNR Cells

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Cell phenotype, transduction efficiency, activation, and exhaustion of TGF-β DNR transduced cells and their nontransduced counterparts were determined by flow cytometry, using the following cell surface markers: CD3, CD56 (BioLegend, San Diego, CA), TGF-β RII (“wildtype” R&D, Minneapolis, MN), TGF-β RII (“DNR” Cambridge, UK), goat-anti mouse IgG, CD16, NKG2D, DNAM-1, NKp30, NKp46, CCR2, and CX3CR1 (BioLegend, San Diego, CA and BD Biosciencees, Franklin Lakes, NJ). Where reported, MFI was calculated from the geometric mean.

Powell A.B., Yadavilli S., Saunders D., Van Pelt S., Chorvinsky E., Burga R.A., Albihani S., Hanley P.J., Xu Z., Pei Y., Yvon E.S., Hwang E.I., Bollard C.M., Nazarian J, & Cruz C.R. (2019). Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization. Journal of Translational Medicine, 17, 321.

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Multiparametric Flow Cytometry Assay

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For flow cytometry, fluorochrome-conjugated antibodies against the following antigens were used: CD56 (clone NCAM16.2; conjugated to BV605) (B159; APC), CD3 (SK7; BUV395) (HIT3a; PE), CD107a (H4A3; APC), IFN-γ (B27; PerCP/Cy5.5), TNF (MAb11; PE), CD69 (FN50; BV421 and BV711), CD11a (HI111; BV711), NKG2D (149810; PE-CF594), NKp30 (p30-15; BV421), TACTILE (6F9; BV711), TRAIL (RIK-2; BV421), BTLA (J168-540; PE-CF594), Phospho-Lck (4/LCK-Y505; AF647), Phospho-LAT (J96-1238.58.93; PE), Phospho-SLP-76 (J141-668.36.58; PE), Phospho-MEK1/2 (O24-836; PE), Phospho-ERK1/2 (20A; BV421), Phospho-SHP-2 (L99-921; AF647) (all BD Biosciences), CD160 (BY55; PE/Cy7), NKp46 (9E2; PE/Cy7),NKp44 (P44-8; PE), DNAM-1 (11A8; APC), 2B4 (C1.7; PerCP/Cy5.5), NKp80 (5D12; PE) (all BioLegend), NKG2C (134591; APC) (R&D Systems), and VZV gE:gI (SG1-1; conjugated in-house to DyLight 488) (Meridian Life Science). Matched isotype control antibodies were also used where appropriate.

Campbell T.M., McSharry B.P., Steain M., Russell T.A., Tscharke D.C., Kennedy J.J., Slobedman B, & Abendroth A. (2019). Functional paralysis of human natural killer cells by alphaherpesviruses. PLoS Pathogens, 15(6), e1007784.

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