Human NKG2D/CD314 Antibody (149810)

R&D Systems | Catalog # MAB139

Clone 149810 was used by HLDA to establish CD designation
R&D Systems
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Key Product Details

Species Reactivity

Validated:

Human

Cited:

Human, Mouse

Applications

Validated:

Western Blot, Neutralization, Flow Cytometry, Costimulation of T Cells, CyTOF-ready

Cited:

Immunohistochemistry, Immunohistochemistry-Frozen, Neutralization, Flow Cytometry, Immunocytochemistry, Bioassay, Blocking, Blocking/Neutralizing, Cytotoxicity Assay, ELISA Capture, Functional Assay, Stimulation, Surface Plasmon Resonance

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 Clone # 149810
View all NKG2D/CD314 Primary Antibodies »

Product Specifications

Immunogen

BaF3 mouse pro-B cell line transfected with human NKG2D/CD314

Specificity

Detects human NKG2D/CD314 in Western blots.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1

Endotoxin Level

<0.10 EU per 1 μg of the antibody by the LAL method.

Scientific Data Images for Human NKG2D/CD314 Antibody (149810)

Detection of NKG2D/CD314 antibody in Human PBMCs gated on CD3- cells antibody by Flow Cytometry.

Detection of NKG2D/CD314 in Human PBMCs gated on CD3- cells by Flow Cytometry.

Human peripheral blood mononuclear cells (PBMCs) gated on CD3- cells were stained with Mouse Anti-Human NCAM-1/CD56 APC-conjugated Monoclonal Antibody (Catalog # FAB2408A) and either (A) Mouse Anti-Human NKG2D/CD314 Monoclonal Antibody (Catalog # MAB139) or (B) Mouse IgG1 Isotype Control (Catalog # MAB002) followed by anti-Mouse IgG PE-conjugated Monoclonal Antibody (Catalog # F0102B). View our protocol for Staining Membrane-associated Proteins.

Detection of NKG2D/CD314 in Human PBMCs by Flow Cytometry.

Human peripheral blood mononuclear cells (PBMCs) were stained with Mouse Anti-Human CD8 APC-conjugated Monoclonal Antibody (FAB1509A) and either (A) Mouse Anti-Human NKG2D/CD314 Monoclonal Antibody (Catalog # MAB139) or (B) Mouse IgG1 Isotype Control (MAB002) followed by anti-Mouse IgG PE-conjugated Monoclonal Antibody (F0102B). Staining was performed using our Staining Membrane-associated Proteins protocol.
Detection of Human NKG2D/CD314 by Flow Cytometry

Detection of Human NKG2D/CD314 by Flow Cytometry

Multiple receptors and ligands are involved in NK cell-mediated lysis of activated CD4+ T cells.Role of (A) activating and (B) inhibitory NK receptors in NK cell degranulation. Left column: representative histograms (of n≥3) for surface expression of ligands on activated (thick black line) and resting CD4+ T cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated CD4+ T) and filled histogram (resting CD4+ T). Middle- and right column: NK and CD4+ T cells were activated for 4 days in vitro as described, and co-cultured for 4 hours with 10 ug/mL mAb (or relevant isotype-matched control Ig). Degranulation is shown for CD56dim (middle column) and CD56bright (right column) NK cells. Representative histograms of surface expression of receptors on activated (thick black line) and resting NK cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated NK) and filled histogram (resting NK). * P<0.05, ** P<0.005, *** P<0.001. (C) Sorted IL-2-activated CD56dim and CD56bright NK cells were co-cultured with 51Cr-labeled activated CD4+ T cells in a 51Cr-release assay with human IgG4 isotype control (•) or anti-NKG2A mAb (○). Data represents n = 3 experiments. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/22384114), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of NKG2D/CD314 by Flow Cytometry

Detection of NKG2D/CD314 by Flow Cytometry

CD4+NKG2D+ T cells exert pro-migratory, cytolytic and pro-inflammatory properties.(A) Flow cytometry: The dot plot shows a representative example of NKG2D expression on CD3+CD4+CD8-CD56− T cells derived from the peripheral blood of a healthy donor (HD). The bar graph represents the mean frequency of CD4+NKG2D+ T cells in the peripheral blood of HDs (n = 20), stable RRMS (n = 15) and active RRMS patients (n = 14). (B–E) Mean fluorescence intensity (MFI) of different markers indicative for migratory capacity (B), activation (C), or cytolytic capacity (D, E) of CD4+NKG2D+ and CD4+NKG2D− T cells from the peripheral blood of HDs (n = 6) or active RRMS patients (n = 6). (F) Percentages of naive (CD45RA+CD62L+), T central memory (Tcm, CD45RA-CD62L+), T effector memory (Tem, CD45RA-CD62L−) and T effector memory RA (Tem-RA, CD45RA+CD62L-) cells in the CD4+NKG2D+ and CD4+NKG2D− T cell compartment assessed by flow cytometry (n = 6 HDs). (G) Carboxyfluorescein succinimidyl ester (CFSE) proliferation assays of CD4+NKG2D+ T cells and CD4+NKG2D− T cells under CD3/CD28, CD3/NKG2D, MOG35-55 (10 µg/ml or 100 µg/ml), MBP1-11 or PLP190-209 stimulation (n = 8 HDs). (H) Intracellular cytokine staining for IFN-gamma and IL-17 of CD4+NKG2D+ and CD4+NKG2D− T cells derived from the peripheral blood of HDs (n = 7). The dot plots depict a representative example of IFN-gamma - and IL-17-positive cells upon CD3/CD28 stimulation. The bar graphs show the frequencies of IFN-gamma or IL-17 positive cells of unstimulated, CD3/CD28- or CD3/NKG2D-stimulated cells. (I) Comparison of the proportions of IFN-gamma or IL-17 positive CD3/CD28-stimulated CD4NKG2D+ T cells derived from frozen PBMCs of HDs (n = 6) or active RRMS patients (n = 6). *P < 0.05. ns, not significant; unstim., unstimulated. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/24282598), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of NKG2D/CD314 by Flow Cytometry

Detection of NKG2D/CD314 by Flow Cytometry

NKG2D and LFA-1 ligation trigger morphologically different responses in NK cells. (H) Quantification of f-actin (phalloidin-AlexaFluor633) across the diameter of cells stimulated on anti-NKG2D (solid line) or anti-LFA-1 (dotted line) (horizontal bars indicate p<0.05 by ANOVA, n = 20). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/19636352), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of NKG2D/CD314 by Flow Cytometry

Detection of NKG2D/CD314 by Flow Cytometry

Consequences of MICA-mediated and antibody-mediated NKG2D engagement on cell activation, NKG2D and TIGIT expression. Primary cultured NK cells were incubated with plate-bound recombinant MICA Fc-chimera or anti-NKG2D mAb and goat anti-mouse antibody (GAM). GAM-stimulated NK cells were used as control of NKG2D stimulation. (A) Cells were harvested after 4 hours of stimulation, and degranulation was measured via flow cytometry by assessing CD107a endosomal marker expression. A representative experiment is shown in left panel while means ± SD of three independent experiments are shown in right panel. Paired t test was used for comparisons. *P < 0.05. (B, C) Surface expression of NKG2D and TIGIT receptors was evaluated by FACS analysis upon 18 hours of stimulation. One representative experiment is shown in left panels. Right panels: data pooled from six independent experiments (mean ± SD) are shown (the mean fluorescence intensity, MFI, value of controls NK cells in each experiment is set to 1). Paired t test was used for comparisons. *ρ < 0.05, **ρ < 0.01, ***ρ < 0.001. (D) Relative TIGIT mRNA amount, normalized with GAPDH, was expressed as arbitrary units and referred to the relative controls, considered as calibrator (the value of the calibrator in each run is set to 1). Means ± SD of five independent experiments are shown. Paired t test was used for comparisons. *ρ < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40421025), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of NKG2D/CD314 by Flow Cytometry

Detection of NKG2D/CD314 by Flow Cytometry

NKG2D stimulation impairs DNAM-1-triggered ERK1/2 phosphorylation. (A, B) goat anti-mouse (GAM) and anti-NKG2D-stimulated NK cells were incubated with anti-DNAM-1 mAb or isotype control Ab followed by goat anti-mouse Ab for different lengths of times. Then, cells were fixed and permeabilized, stained with anti-pAKT (panel A) and anti-pERK1/2 (panel B) and analyzed by FACS. Results from 2 minutes of stimulation with anti-DNAM-1 mAb (filled histograms) or isotype control Ab (empty histograms) from one representative of three independent experiments is shown in the left panels. Quantitative changes in protein phosphorylation at the indicated lengths of time of three independent experiments (mean ± SD) are shown in the right panels. The ratio between the mean fluorescence intensity (MFI) of anti-DNAM-1-stimulated and Ctrl-Ig samples for each time point was used to calculate fold changes. Multiple t test was used for comparisons. *ρ < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40421025), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of NKG2D/CD314 by Flow Cytometry

Detection of NKG2D/CD314 by Flow Cytometry

NKG2D internalization is required for impairment in DNAM-1-mediated cytotoxic granule polarization. (A) Surface expression of NKG2D (upper panels) and TIGIT (lower panels) receptors was evaluated on goat anti-mouse (GAM) or anti-NKG2D-stimulated DAP10 WT and DAP10K84R NKL. One representative of three independent experiments is shown in left panels. Data pooled from three independent experiments (mean ± SD) are shown in right panels (the mean fluorescence intensity, MFI value of GAM-stimulated cells in each experiment is set to 1). Paired t test was used for comparisons. *ρ < 0.05. (B) GAM and anti-NKG2D-stimulated DAP10 WT and DAP10K84R NKL (effector cells, E) were incubated with Ba/F3-PVR (target cells, T) for 30 minutes, fixed, permeabilized and stained with the anti-Perforin mAb followed by AlexaFluor594-conjugated GAM-IgG2b and AlexaFluor488-conjugated Phalloidin to visualize polarized actin. Cells were then counterstained with DAPI to visualize nuclei and acquired with LSM980 confocal microscope with a 63x/1.4 NA UPlanSAPO oil immersion objective (all from Zeiss). Right panel: Percentages of conjugates with polarized granules are shown as mean ± SD calculated on ≥30 conjugates for each NK cell type of three independent experiments. Paired t test was used for comparisons. *ρ < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40421025), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of NKG2D/CD314 by Flow Cytometry

Detection of NKG2D/CD314 by Flow Cytometry

NKG2D stimulation impairs DNAM-1-triggered ERK1/2 phosphorylation. (A, B) goat anti-mouse (GAM) and anti-NKG2D-stimulated NK cells were incubated with anti-DNAM-1 mAb or isotype control Ab followed by goat anti-mouse Ab for different lengths of times. Then, cells were fixed and permeabilized, stained with anti-pAKT (panel A) and anti-pERK1/2 (panel B) and analyzed by FACS. Results from 2 minutes of stimulation with anti-DNAM-1 mAb (filled histograms) or isotype control Ab (empty histograms) from one representative of three independent experiments is shown in the left panels. Quantitative changes in protein phosphorylation at the indicated lengths of time of three independent experiments (mean ± SD) are shown in the right panels. The ratio between the mean fluorescence intensity (MFI) of anti-DNAM-1-stimulated and Ctrl-Ig samples for each time point was used to calculate fold changes. Multiple t test was used for comparisons. *ρ < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40421025), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of NKG2D/CD314 by Immunocytochemistry/ Immunofluorescence

Detection of NKG2D/CD314 by Immunocytochemistry/ Immunofluorescence

NKG2D internalization is required for impairment in DNAM-1-mediated cytotoxic granule polarization. (A) Surface expression of NKG2D (upper panels) and TIGIT (lower panels) receptors was evaluated on goat anti-mouse (GAM) or anti-NKG2D-stimulated DAP10 WT and DAP10K84R NKL. One representative of three independent experiments is shown in left panels. Data pooled from three independent experiments (mean ± SD) are shown in right panels (the mean fluorescence intensity, MFI value of GAM-stimulated cells in each experiment is set to 1). Paired t test was used for comparisons. *ρ < 0.05. (B) GAM and anti-NKG2D-stimulated DAP10 WT and DAP10K84R NKL (effector cells, E) were incubated with Ba/F3-PVR (target cells, T) for 30 minutes, fixed, permeabilized and stained with the anti-Perforin mAb followed by AlexaFluor594-conjugated GAM-IgG2b and AlexaFluor488-conjugated Phalloidin to visualize polarized actin. Cells were then counterstained with DAPI to visualize nuclei and acquired with LSM980 confocal microscope with a 63x/1.4 NA UPlanSAPO oil immersion objective (all from Zeiss). Right panel: Percentages of conjugates with polarized granules are shown as mean ± SD calculated on ≥30 conjugates for each NK cell type of three independent experiments. Paired t test was used for comparisons. *ρ < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40421025), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human NKG2D/CD314 Antibody (149810)

Application
Recommended Usage

Costimulation of T Cells

Rincon-Orozco, B. et al. (2005) J. Immunol. 175:2144.

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Flow Cytometry

0.25 µg/106 cells
Sample: Human PBMC

Western Blot

1 µg/mL
Sample: Recombinant Human NKG2D/CD314 Fc Chimera (Catalog # 1299-NK)
under non-reducing conditions only

Neutralization

Kwong, K.Y. et al. (2008) J. Mol. Biol. 384:1143.

Reviewed Applications

Read 3 reviews rated 5 using MAB139 in the following applications:

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Protein A or G purified from hybridoma culture supernatant

Reconstitution

Reconstitute at 0.5 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: NKG2D/CD314

NKG2D, also known as CD314, is a type II transmembrane glycoprotein having an extracellular lectin-like domain. This domain lacks the recognizable calcium-binding sites found in true C‑type lectins and binds protein rather than carbohydrate ligands. Human NKG2D is expressed on CD8+ alpha beta T cells, gamma delta T cells, NK cells, and NKT cells. In mouse systems NKG2D also occurs on macrophages. Human ligands for NKG2D include MICA, MICB, and ULBP1, 2, and 3. Expression of NKG2D ligands occurs in epithelial cells, tumor cells and under conditions of stress or infection. NKG2D exists as a disulfide-linked homodimer that delivers an activating signal upon ligand binding. Signaling requires association with an adapter protein. Alternative splicing of the NKG2D mRNA results in isoforms with different cytoplasmic domains that can associate either with DAP12 to deliver a true activating signal or with DAP10 resulting in a costimulatory signal. NKG2D has been implicated in anti-tumor surveillance and the immune response against viral infection.

References

  1. Li, P. et al. (2001) Nature Immunol. 2:443.
  2. Steinle, A. et al. (2001) Immunogenetics 53:279.
  3. Cosman, D. et al. (2001) Immunity 14:123.
  4. Cerwenka, A. and L. Lanier (2001) Immunol. Rev. 181:158.
  5. Wu, J. et al. (1999) Science 285:730.
  6. Diefenbach, A. et al. (2002) Nature Immunol. 3:1142.
  7. Gilfillan, S. et al. (2002) Nature Immunol. 3:1150.
  8. Groh, V. et al. (2001) Nature Immunol. 2:255.
  9. Cerwenka, A. et al. (2001) Proc. Natl. Acad. Sci. USA 98:11521.
  10. Diefenbach, A. et al. (2001) Nature 413:165.
  11. NKG2D and its Ligands (2002) www.RnDSystems.com.

Long Name

Natural Killer G2D

Alternate Names

CD314, D12S2489E, KLRK1

Entrez Gene IDs

22914 (Human); 27007 (Mouse); 102120479 (Cynomolgus Monkey)

Gene Symbol

KLRK1

Additional NKG2D/CD314 Products

Product Documents for Human NKG2D/CD314 Antibody (149810)

Certificate of Analysis

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Product Specific Notices for Human NKG2D/CD314 Antibody (149810)

For research use only

Citations for Human NKG2D/CD314 Antibody (149810)

Customer Reviews for Human NKG2D/CD314 Antibody (149810) (3)

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Showing  1 - 3 的 3 reviews Showing All
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  • Human NKG2D/CD314 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: Peripheral blood mononuclear cells (PBMCs)
    Species: Human
    Verified Customer | Posted 01/28/2022
    Peripheral blood mononuclear cells (PBMCs)
    Human NKG2D/CD314 Antibody (149810) MAB139
  • Human NKG2D/CD314 Antibody
    Name: Anonymous
    Application: Flow Cytometry
    Sample Tested: Peripheral blood mononuclear cells (PBMCs)
    Species: Human
    Verified Customer | Posted 01/24/2021
    Human NKG2D/CD314 Antibody (149810) MAB139
  • Human NKG2D/CD314 Antibody
    Name: Leslie Priddy
    Application: Immunohistochemistry
    Sample Tested: Paraffined tumor tissue from HCC xenograft
    Species: Human
    Verified Customer | Posted 04/13/2018

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