Nkg2d apc

Manufactured by BD

Sourced in United States

NKG2D-APC is a flow cytometry reagent used to detect the expression of NKG2D receptor on immune cells. NKG2D is a key activating receptor expressed on natural killer cells, CD8+ T cells, and other lymphocyte populations. The APC fluorophore is conjugated to the NKG2D antibody, allowing for the detection and quantification of NKG2D-positive cells by flow cytometry.

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Lab products found in correlation

Nkg2a pe, by Beckman Coulter (4 mentions) Cd56 pe cy7, by BD (3 mentions) Fixable blue dead cell stain, by Thermo Fisher Scientific (3 mentions) Kir3dl1 alexafluor700, by BioLegend (3 mentions) Cd94 fitc, by BD (3 mentions) 2b4 fitc, by BD (3 mentions) Nkp46 pe, by BD (3 mentions) Nkp30 apc, by BD (3 mentions) Trail pe, by BioLegend (3 mentions) Cd161 fitc, by Miltenyi Biotec (3 mentions) Cd160 alexafluor647, by BioLegend (3 mentions) Cd158a percp cy5, by Thermo Fisher Scientific (3 mentions) Cd3 percp, by BD (3 mentions) Facscalibur, by BD (3 mentions) Cd56 pe, by BD (2 mentions) Cd3 fitc, by BD (2 mentions) Cd27 percp cy5, by BD (2 mentions) Cd56 fitc, by BD (2 mentions) Cd158b fitc, by BD (2 mentions) Anti perforin percp cy5, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

Anti-NKG2D-APC (7 citations)

17 protocols using nkg2d apc

Multiparameter Characterization of NK Cells

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PBMCs or tissue-infiltrating lymphocytes were stained as follows. Frozen cells were thawed and resuspended in complete RPMI1640 medium (Corning, 10–040-CVR) containing 10% fetal bovine serum (Gibco, 12662029), 1% glutamine (Immundiagnostic, K7732) and 1% penicillin and streptomycin (Gbico, 15140–122). 1×10⁶ cells were used for each panel and stained in dark at room temperature (20–25 °C) for 30 min. The following monoclonal antibodies (Abs) were used for NK cell phenotypic characterization: Pacific Blue-Vivid (Invitrogen, CA), PE-CF594-CD3 (BD Biosciences, 562280), Alexa Fluor® 700-CD14 (BD Biosciences, 557923), PE-Cy7-CD56 (BD Biosciences, 335791), APC-NKG2D (BD Biosciences, 558071), PE-NKG2C (R&D, FAB138C), PE-NKG2A (R&D, FAB1059C), APC-CD69 (BD Biosciences, 340560), PerCP-Cy5-HLA-DR (BD Biosciences, 347364) and FITC-CD38 (BD Biosciences, 555459). After staining, the PBMCs were washed twice with phosphate buffer saline and detected using a BD LSR II Fortessa flow cytometer (BD Biosciences, NJ). The data were analyzed using the FlowJo software (TreeStar, San Carlos, CA).

Gao J., Duan Z., Zhang L., Huang X., Long L., Tu J., Liang H., Zhang Y., Shen T, & Lu F. (2015). Failure recovery of circulating NKG2D+CD56dimNK cells in HBV-associated hepatocellular carcinoma after hepatectomy predicts early recurrence. Oncoimmunology, 5(1), e1048061.

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Analyzing NK Cell Cytokine Production

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Purified NKG2D⁺NK cells from eight HCC patients and eight HD were sorted by flow cytometry (BD, FACSAriaII) and stimulated with PMA (25 ng/mL) (Sigma-Aldrich, P1585) and Ionomycin (1 μg/mL) (Santa Cruz Biotechnology, 56092-82-1) at 37°C for 1 h. Then, Pecy5-CD107a (BD Biosciences, 555802), GolgiStop (BD Biosciences, 554715) and Brefedlin A (Santa Cruz Biotechnology, sc-200861) were added into the medium. After 5 h, cells were collected and stained with the following monoclonal surface Abs, eFluor®450-CD3 (eBioscience, 48-0038-42), PE-CD14 (BD Biosciences, 555398), Pecy7-CD56 (BD Biosciences, 335791), APC-NKG2D (BD Biosciences, 558071). Then, cells were intracellularly stained with Alexa Fluor® 700-IFNγ (BD Biosciences, 557995) and FITC-TNF-α (BD Biosciences, 554512) and detected by flow cytometry.

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Phenotyping of Immune Cells by Flow Cytometry

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Antibodies for surface markers staining of DCs and T cells were obtained from BD Biosciences, New York, USA (anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC, CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC, CD27-PerCPCY5.5, CD57-APC, CCR7-PE, CD14-APC, CD80-PE, CD83-APC, CD86-FITC, HLA-DR-FITC). Antibodies for intracellular proteins staining were also obtained from BD Biosciences (anti-human IFNγ-APC, TNFα-PECY7, granzyme B-FITC, FoxP3-PE). The intracellular staining was performed by fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences). The experiments were performed by using FACS CantoII (BD Biosciences) flow cytometer, and data were analyzed by using the Flowjo software.

Han Y., Wu Y., Yang C., Huang J., Guo Y., Liu L., Chen P., Wu D., Liu J., Li J., Zhou X, & Hou J. (2017). Dynamic and specific immune responses against multiple tumor antigens were elicited in patients with hepatocellular carcinoma after cell-based immunotherapy. Journal of Translational Medicine, 15, 64.

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Comprehensive NK Cell Profiling in Melanoma

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NK cell phenotype of melanoma patients enrolled in the trial was examined using fluorochrome-conjugated antibodies against the following cell-surface markers: CD56-FITC, CD3-PC7, CD16-APC, CD69-BV421, NKp30-BV711, CXCR3-BV421, CCR3-BV510 (BD Biosciences; San Diego, CA), NKp44-PerCP eFluor 710 (eBioscience; San Diego, CA), CXCR1-PE (R&D Systems; Minneapolis, MN), CCR7-BV711 (BioLegend; San Diego, CA), and matching IgG isotype controls from the same vendors. The immune checkpoint and NK cell activation receptor panel included the following markers: Zombie NIR Fixable Viability Dye (BioLegend; San Diego, CA), CD3-PE-Vio770 (Miltenyi Biotec; San Diego, CA), ANK-1-PE (Santa Cruz Biotechnology; Dallas, TX), TIGIT-PerCP eFluor 710 (eBioscience), CD45-BUV395, CD56-BV510, CD16-BUV737, NKG2D-APC, NKp46-BV711, CD69-BV421, and PD-1-BV650 (BD Biosciences).

Vujanovic L., Chuckran C., Lin Y., Ding F., Sander C.A., Santos P.M., Lohr J., Mashadi-Hossein A., Warren S., White A., Huang A., Kirkwood J.M, & Butterfield L.H. (2019). CD56dim CD16− Natural Killer Cell Profiling in Melanoma Patients Receiving a Cancer Vaccine and Interferon-α. Frontiers in Immunology, 10, 14.

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Surface Marker Staining of γδTc

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For surface marker staining of γδTc, the following anti-human antibodies from BioLegend, unless otherwise indicated, were employed: TCRγδ PE (clone B1, 1:25); TCR Vδ1 FITC (Miltenyi, clone REA173, 1:10); TCR Vδ2 PerCP (clone B6, 1:25); NKG2D APC (BD Biosciences, Mississauga, ON, Canada, 1:25); CD56 FITC (clone MEM-188, 1:5); CD69 AF700 (clone FN50, 1:4); CD94 FITC (clone DX22, 1:5); CD95 APC (clone DX2, 1:100); HLA ABC PE (clone W6/32, 1:10); FasL PE (clone NOK-1, 1:5); and CD40L APC (clone 24–31, 1:5).
Anti-human MICA/B PE (BioLegend, clone 6D4, 0.1 µg) was used to stain breast cancer cell lines.

Siegers G.M., Dutta I., Lai R, & Postovit L.M. (2018). Functional Plasticity of Gamma Delta T Cells and Breast Tumor Targets in Hypoxia. Frontiers in Immunology, 9, 1367.

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Comprehensive NK Cell Phenotyping

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Flow cytometry was performed on cryopreserved cells. As per the gating strategy in figure 1A, NK cells were identified using a combination of Fixable blue dead cell stain (Life technologies), CD3-Pacific Blue, CD56-PE-Cy7 and CD16- APC-Cy7 (BD Biosciences). NK cell receptor expression was assessed using combinations of CD158a-PerCP-Cy5.5 (eBioscience), CD158b-FITC, KIR3DL1-Alexafluor700 (both Biolegend), KIR2DL3-PE, KIR2DL1-APC (both R&D Systems), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). For intracellular staining, cells were fixed and permeabilized (PermA/B solution, Caltag) according to the manufacturers' instructions, prior to incubation with Perforin-PerCP-Cy5.5 (eBioscience). At least 1500 NK cells were acquired for all samples on either a five laser BD LSRFortessa or a four laser BD LSRII system, equipped with FACSDiva Version 8.8.3 (BD biosciences). Rainbow beads ensured a consistent, comparable level of fluorescence across all samples on different days of acquisition. Gates were set using fluorescence minus one or unstimulated samples where appropriate. The data were analysed using FlowJo version 9.5.3 (Treestar, OR, USA).

Cosgrove C., Berger C.T., Kroy D.C., Cheney P.C., Ghebremichael M., Aneja J., Tomlinson M., Kim A.Y., Lauer G.M, & Alter G. (2014). Chronic HCV Infection Affects the NK Cell Phenotype in the Blood More than in the Liver. PLoS ONE, 9(8), e105950.

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Characterization of NK Cell Receptor Expression

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NK cells were incubated with optimal concentrations of the following anti-human Abs: CD56-PE, CD3-PerCP, NKG2D-APC, CD226-FITC, CD96-PE or the corresponding isotype control Abs (all from BD Pharmingen) in 100 μl of 1% FCS/PBS at 4°C for 20 min.
Tumour cells, with and without platelets, were incubated with anti-human MICA, MICB unconjugated antibodies and alexa-fluor-488 secondary antibody. Staining of tumour cells using primary and secondary antibodies was performed for 1 hour at 4°C. Tumour cells were also stained with CD112-APC and CD155-FITC antibodies and isotype controls in 100 μl of 1% FCS/PBS at 4°C for 20 min.
Cells were washed twice with PBS and acquired on a Cyan flow cytometer (Beckman Coulter, Brea, CA, USA). Events were stored and analysed on the FlowJo software (TreeStar). NK cells were gated from PBMC populations as distinct CD3-CD56+ cells (S1 Fig).

Cluxton C.D., Spillane C., O'Toole S.A., Sheils O., Gardiner C.M, & O'Leary J.J. (2019). Suppression of Natural Killer cell NKG2D and CD226 anti-tumour cascades by platelet cloaked cancer cells: Implications for the metastatic cascade. PLoS ONE, 14(3), e0211538.

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Comprehensive NK-Cell Receptor Profiling

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Cryopreserved peripheral blood mononuclear cells (PBMC) were stained with combinations of antibodies that comprehensively interrogate the breadth of NK-cell receptors including the killer immunoglobulin receptors (KIRs), the c-type lectin receptors (NKG2), and the natural cytotoxicity receptors (NCRs). These included CD158a-PerCP-Cy5.5 (eBioscience), KIR3DL1-Alexafluor700 (Biolegend), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC, CD158b-FITC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). Dead cells were excluded using the Fixable Blue Dead Cell Stain (Life Technologies) prior to surface staining and fixation. For perforin staining, samples were then permeabilized (Perm A/B, Caltag) and stained with anti-Perforin-PerCP-Cy5.5 (eBioscience). NK cells were identified as CD3 negative lymphocytes expressing CD56 and/or CD16. At least 1500 NK cells were acquired per sample. Fluorescence minus one was used to set gates. The data were analyzed with Flowjo v9.5.4 (Treestar).

Yu W.H., Cosgrove C., Berger C.T., Cheney P.C., Krykbaeva M., Kim A.Y., Lewis-Ximenez L., Lauer G.M, & Alter G. (2018). ADCC-Mediated CD56DIM NK Cell Responses Are Associated with Early HBsAg Clearance in Acute HBV Infection. Pathogens & Immunity, 3(1), 2-18.

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Immunophenotyping of T-cell subsets in PBMC

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Human peripheral blood samples were collected in 4 heparin tubes at baseline, following nivolumab infusion and multiple timepoints following TIL infusion. Peripheral blood mononuclear cells (PBMC) were collected using a Ficoll gradient and cryopreserved in 10% DMSO and FBS. Cells were thawed in media, and subsequently stained in PBS containing 5% FBS (vol/vol, FACS buffer) with: CD3 BUV496, CD56 BUV563, CD4 BUV737, CD197 BV421, CD28 BV480, CD14 BV605, CD19 BV605, CD95 BV711, CD195 BV786, CD127 PE, CD194 PE-Cy7, CD45RA Alexa488, CD25 PerCP-Cy5.5, NKG2D APC, Tim3 BV421, PD1 BV480, CD226 BV711, CTLA4 BV786, Lag3 PE, TIGIT PE-Cy7, CD244 Alexa488, CD27 PerCP-Cy5.5, BTLA APC from BD Biosciences. Dead cells were excluded using the Zombie NIR Fixable Viability Kit from Biolegend, incubated at 4 °C for 1 hr, then washed twice with FACS buffer, and finally fixed in PBS containing 1% paraformaldehyde before running flow cytometry. Cells were acquired on a BD FACSymphony™ A5, and data were analyzed with FlowJo Version 10.0 software. All cell gates were drawn uniformly for analysis across patients and time points, with gating strategy provided in Supplementary Appendix 2. Plots of t-SNE were generated by Flowjo Version 10.6.1 according to the expression of CD45RA, CCR7, CD28 and CD95. Different memory T cell subsets were shown using separate colors.

Creelan B.C., Wang C., Teer J.K., Toloza E.M., Yao J., Kim S., Landin A.M., Mullinax J.E., Saller J.J., Saltos A.N., Noyes D.R., Montoya L.B., Curry W., Pilon-Thomas S.A., Chiappori A.A., Tanvetyanon T., Kaye F.J., Thompson Z.J., Yoder S.J., Fang B., Koomen J.M., Sarnaik A.A., Chen D.T., Conejo-Garcia J.R., Haura E.B, & Antonia S.J. (2021). Tumor-infiltrating lymphocyte treatment for anti-PD-1 resistant metastatic lung cancer: a phase I trial. Nature medicine, 27(8), 1410-1418.

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Cell Surface Receptor Analysis

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For analysis of surface receptors, 1 × 10⁶ cells of each cell line were stained with 5 µl anti-human PE-conjugated antibody to MICA/B (eBioscience; clone 6D4) or 2.5 uL PE-conjugated antibody to HLA-ABC (BD: Clone W6/32)Following incubation with anti-MICA/B antibody or anti-HLA-ABC, cells were washed and resuspended in FACs buffer (1X PBS containing 2% BSA and 0.1% sodium azide) and acquired on a LSR II Flow Cytometer (BD Biosciences). Primary CTLs were stained with the following antibodies: CD8-FITC (BD Pharmingen; clone SK1), CD3-PE (Biolegend; clone SK7), NKp44-APC (R&D Systems; clone 253415), NKp46-APC (BD Pharmingen; clone 9-E2), KIR2DS4-APC (R&D Systems; clone 179315), NKG2C-APC (R&D Systems; clone 134591), and NKG2D-APC (BD Pharmingen; clone 1D11). Data were analyzed using FlowJo version 10 and figures were prepared in GraphPad Prism 7.

Trinh T.L., Kandell W.M., Donatelli S.S., Tu N., Tejera M.M., Gilvary D.L., Eksioglu E.A., Burnette A., Adams W.A., Liu J., Teer J.K., Djeu J.Y., Coppola D, & Wei S. (2019). Immune evasion by TGFβ-induced miR-183 repression of MICA/B expression in human lung tumor cells. Oncoimmunology, 8(4), e1557372.

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