Nkg2d apc

Manufactured by Miltenyi Biotec

Sourced in Germany, France, United States

NKG2D-APC is a monoclonal antibody conjugated with the fluorescent dye APC (allophycocyanin). It is designed for the detection and analysis of NKG2D-expressing cells, such as natural killer (NK) cells and certain T cell subsets, using flow cytometry.

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5 protocols using nkg2d apc

Comprehensive Immune Profiling of Cohorts

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MLs phenotypes from patients’ samples of the ELYP cohort at different timepoints were performed using CD103-FITC, CD73-PE, CTLA4-APC, CD39-APC-Vio770, CCR6-PE, PD1-PE-Vio770, NKG2D-APC, CD45RO-APC-Vio770 (Miltenyi), CD3-AF700, CD161-PE-Cy5, CD8-BV605 and CD4-BV711 (BD Biosciences), . MLs phenotypes from patients’ samples of the REMIND and IMCO cohort at different timepoints were performed using CD69-FITC, CD45RO-PerCP, CD5-AF700, CD8-BV605, CD4-BV711 (BD Biosciences), NKG2D-APC and CD103-APC-Vio770 (Miltenyi). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in FACS buffer (Miltenyi).
For the allogenic cocultures, cells were then stained using the following antibodies: Annexin V-FITC, E-Cadherin-PE-Vio770 HLA-E-APC, MICA/MICB-APC-Vio770 (Miltenyi), HLA-ABC-AF700 (BioLegend), HLA-DPDQDR-BV510, CD45-BV605 (BD Biosciences) and ULBP 2/5/6-PE (RD). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in Annexin V binding buffer (Miltenyi).

Hammoudi N., Hamoudi S., Bonnereau J., Bottois H., Pérez K., Bezault M., Hassid D., Chardiny V., Grand C., Gergaud B., Bonnet J., Chedouba L., Tran Minh M.L., Gornet J.M., Baudry C., Corte H., Maggiori L., Toubert A., McBride J., Brochier C., Neighbors M., Le Bourhis L, & Allez M. (2022). Autologous organoid co-culture model reveals T cell-driven epithelial cell death in Crohn’s Disease. Frontiers in Immunology, 13, 1008456.

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Characterizing Mouse and Human NK Cell Populations

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The following Abs against mouse molecules were purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany): CD3-fluorescein isothiocyanate (FITC), NK1.1-peridine-chlorophyll-protein (PerCP)-Vio700, CD11b-allophycocyanin (APC)-Vio770, CD27-phycoerythrin (PE)-Vio770, NKG2D-APC, NKp46-PE-Vio770, KLRG1-PE, Ly49D-PE, CD4-PerCP-Vio700 and CD8a-APC for mouse molecules, and CD56-FITC, CD107a-PE and IFN-γ-APC for human molecules. We purchased 7-amino-actinomycin D (7-ADD) and FITC-conjugated annexin V from BD Biosciences (San Jose, CA, USA). Flow cytometric analysis was performed using a Cytoflex flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and data analyses were performed using the CytExpert software (Beckman Coulter, Inc.).

Jang S.H., Oh M.S., Baek H.I., Ha K.C., Lee J.Y, & Jang Y.S. (2019). Silk peptide treatment potentiates natural killer cell activity in vitro and induces natural killer cell maturation and activation in mouse splenocytes. Pharmaceutical Biology, 57(1), 369-379.

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Isolation and Characterization of Human NK Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stored in liquid nitrogen. NK cell purification was performed with the Dynabeads Untouched Human NK cells kit (Life Technologies, Paisley, UK). Antibodies used were: CD56-PECy7, CD3-PerCP, CD16-APC-Cy7, CD57-Pacific Blue (all BioLegend, San Diego, USA), CD56-fluorescein isothiocyanate (FITC) (BD Biosciences, Oxford, UK), CD3-Pacific Blue (eBioscience, Hatfield, UK), CD158a,h-PE, CD158b-FITC, CD158b1/b2,j-PE (all Beckman Coulter, Marseille, France), NKp30-APC, NKp46-APC, NKG2D-APC (all Miltenyi Biotec, Gladbach, Germany), NKG2C-PE, NKG2C-PErCP, NKG2C-APC (all R&D Systems Europe, Oxford, UK), rabbit STAT4 (Invitrogen, Paisley, UK) with goat anti rabbit secondary-APC (Abcam, Cambridge, UK).

Jamil K.M., Hydes T.J., Cheent K.S., Cassidy S.A., Traherne J.A., Jayaraman J., Trowsdale J., Alexander G.J., Little A.M., McFarlane H., Heneghan M.A., Purbhoo M.A, & Khakoo S.I. (2016). STAT4-associated natural killer cell tolerance following liver transplantation. Gut, 66(2), 352-361.

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NK Cell Surface Marker Profiling

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NK3.3-LTV cells were analyzed for changes in cell surface marker expression compared to non-immortalized NK3.3 cells. All cells (1 x 10⁵ cells per antibody) were centrifuged at 300 g x 5 min, media aspirated, and 1.5 µL single antibody + 20 µL staining buffer (phosphate buffered saline (PBS) + 1% bovine serum albumin + 1% sodium azide) were added to cells. Sample tubes were incubated for 30 min on ice, followed by addition of 200 µL staining buffer and incubated for another 10 min on ice. Samples were analyzed using LSRFortessa Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using FlowJo software. Antibodies used include: NKp46 PE/Cy7 (#331915, BioLegend, San Diego, CA); NKp30 PE (#325207, BioLegend); NKp44 PE (#IM3710, Beckman Coulter, Brea, CA); CD94 PE (#130-098-974, Miltenyi Biotec, Auburn, CA); NKG2D APC (#120-003-706, Miltenyi Biotec); CD161 APC-Alexa Fluor750 (#B30630, Beckman Coulter); CD158e1 BV421 (#312713, BioLegend); CD158a,h PE/Cy7 (#A66899, Beckman Coulter); CD244 PerCp Cy5.5 (#B21171, Beckman Coulter); CD16 ECD (#A33098, Beckman Coulter); CD133 APC (#130-098-829, Miltenyi Biotec); CD57 BV421 (#563896, BD Biosciences); CD158 FITC (#339503, BioLegend).

Matchett E.C, & Kornbluth J. (2023). Extracellular vesicles derived from immortalized human natural killer cell line NK3.3 as a novel therapeutic for multiple myeloma. Frontiers in Immunology, 14, 1265101.

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Multiparametric NK Cell Phenotyping

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Single-cell suspensions from blood, omentum, and liver were stained with CD3-APC-Cy7 (BioLegend, San Diego, CA, USA) and CD56-APC, CD56-FITC, NKP46-PE, NKP30-APC, NKG2D-APC, CCR5-FITC, CXCR3-PE, CCR1-APC, CCR2-APC, CXCR2-FITC, CCR3-PE, and CCR6-APC (Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells were quantified as CD56 + CD3 2 cells within the lymphocyte gate. For cytokine profiling and degranulation assay, cells were stimulated with PMA (10 ng/ml) and ionomycin (1 mg/ml) for 1 h. PE-conjugated anti-CD107a (BD Biosciences UK, Oxford, United Kingdom) was added at this time to detect degranulation, followed by the addition of brefeldin A (10 mg/ml) for a further 3 h. Subsequent staining with IFNg-V500, TNF-a-APC, and IL-10-PE (BD Biosciences UK) was performed. Cells were acquired using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Conroy M.J., Fitzgerald V., Doyle S.L., Channon S., Useckaite Z., Gilmartin N., O'Farrelly C., Ravi N., Reynolds J.V, & Lysaght J. (2016). The microenvironment of visceral adipose tissue and liver alter natural killer cell viability and function. Journal of leukocyte biology, 100(6).

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