Nkg2d 1d11

Manufactured by BioLegend

Sourced in United States

NKG2D (1D11) is a mouse monoclonal antibody that recognizes the natural killer group 2, member D (NKG2D) receptor expressed on natural killer cells, CD8+ T cells, and other immune cells. It is a useful tool for the study of NKG2D and its role in immune cell function.

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Lab products found in correlation

Rab27a, by Abcam (2 mentions) Ci mpr 2g11, by Abcam (2 mentions) Eea 1 14 eea1, by BD (2 mentions) Lamp2 alexafluor 488 h4b4, by Thermo Fisher Scientific (2 mentions) Perforin pf 344, by Mabtech (2 mentions) Nkp46 9e2, by BioLegend (2 mentions) Pkh26 fluorescent dye, by Merck Group (1 mentions) Tnfα mab11, by BioLegend (1 mentions) Dnam 1 dx11, by BD (1 mentions) Rik 2, by BioLegend (1 mentions) Anti mouse cd45 30 f11, by BD (1 mentions) Robosep buffer, by STEMCELL (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Facsaria 2, by BD (1 mentions) Facsdiva, by BD (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Anti tnf α, by Thermo Fisher Scientific (1 mentions) Anti foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions) Anti pstat1 tyr701 58d6, by Cell Signaling Technology (1 mentions) Anti cd45.1 a20, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Anti-NKG2D (1D11) (5 citations) Anti-NKG2D (clone 1D11, (3 citations) Anti-human-NKG2D (1D11) (2 citations) NKG2D blocking antibody (clone 1D11; (2 citations) Anti-NKG2D-APC mAb (clone 1D11, (2 citations)

7 protocols using nkg2d 1d11

In Vitro Cytotoxicity Assay of EV-DNTs and CAR4-DNTs

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Target cells were labeled with 2 µM PKH26 fluorescent dye (Sigma) according to the manufacturer’s instruction and cocultured with EV-DNTs and CAR4-DNTs in a 96-well plate at various effector-to-target ratios for 2 hours or 24 hours at 37°C. Subsequently, cells were stained with annexin V, and apoptosis of the target cells was analyzed by flow cytometry. Per cent specific killing was calculated using the formula:

\frac{% A n n e x i n V + w i t h D N T - % A n n e x i n V + w i t h o u t D N T}{100 - % A n n e x i n V + w i t h o u t D N T} \times 100

as previously described. For transwell assays, an HTS Transwell 96-well permeable support (Sigma) with 0.4 µm pore size was used. For blocking assays, anti-CD18 (TS1/18, BioLegend), NKG2D (1D11, BioLegend), DNAM-1 (DX11, BD Bioscience), TNF-α (MAb11, BioLegend), IFN-γ (MD-1, BioLegend), TRAIL (RIK-2, BioLegend), and FasL (NOK-1, BioLegend) antibodies or immunoglobulin 1 (IgG1) isotype control (QA16A12, BioLegend) was added at 10 µg/mL. For blocking assays with concanamycin A (CMA), EV-DNTs or CAR4-DNTs were treated with CMA (100 nM; Sigma) or dimethyl sulfoxide (DMSO) for 30 min prior to use for in vitro cytotoxicity assay.

Fang K.K., Lee J., Khatri I., Na Y, & Zhang L. (2023). Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells. Journal for Immunotherapy of Cancer, 11(9), e007277.

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Multiplex Imaging of Cytotoxic Granule Components

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The following Abs were used against perforin (dG9), LAMP1-AlexaFluor 488 (H4A3), CD3 (UCHT1), CD56 (HCD56), NKG2D (1D11) (BioLegend); CI-MPR (2G11), dynein heavy chain, Rab27a (Abcam); EEA-1 (14/EEA1; BD Biosciences); cathepsin D, Rab7, Rab9 (Cell Signaling); LAMP1 (H4A3), LAMP2 (H4B4), granzyme B (GB7), Munc13-4, Rab14 (D-5) (Santa Cruz Biotechnology); myosin IIA, actin (AC-15) (Sigma), VAMP7 (Synaptic Systems); LAMP2-AlexaFluor 488 (H4B4) (eBiosciences); perforin (Pf-344, for immunoblotting; Mabtech).

Gil-Krzewska A., Saeed M.B., Oszmiana A., Fischer E.R., Lagrue K., Gahl W.A., Introne W.J., Coligan J.E., Davis D.M, & Krzewski K. (2018). An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome. The Journal of Allergy and Clinical Immunology, 142(3), 914-927.e6.

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Multiparametric Flow Cytometry Panel

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The following antibodies from BioLegend were used for flow cytometry: anti-human CD2 (TS1/8), CD3 (HIT3a), CD10 (HI10a), CD33 (WM53), CD34 (581), CD36 (5–271), CD38 (HB-7), CD45 (H130), CD45RA (HI100), CD56 (HCD56), CD94 (DX22), CD122 (TU27), CD132 (TUGh4), CD135 (BV10A4H2), NKG2D (1D11) and NKp46 (9E2). Anti-human NKG2A antibody was from R&D systems (Minneapolis, MN). Anti-mouse CD45 (30-F11) was acquired from BD Biosciences. For flow cytometry, samples were stained with antibodies in 50 μl flow buffer (0.2% bovine serum albumin [BSA, sigma], 0.05% sodium azide [sigma] in PBS) on ice for 30 mins. Stained samples were then analyzed on a BD LSR II flow cytometer, and data analyzed with FACS Diva (BD Biosciences) and FlowJo (TreeStar version e.g. 7.6.5). Isotype-matched control antibodies were used for all fluorochrome-isotype combinations. For fluorescent-activated cell sorting (FACS), cells were stained with appropriate antibodies in RoboSep buffer (Stemcell Technologies), and sorted on a BD FACSAria II (BD Bioscience).

Chen Q., Ye W., Jian Tan W., Mei Yong K.S., Liu M., Qi Tan S., Loh E., TE Chang K., Chye Tan T., Preiser P.R, & Chen J. (2015). Delineation of Natural Killer Cell Differentiation from Myeloid Progenitors in Human. Scientific Reports, 5, 15118.

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Comprehensive Antibody Panel for Murine Cell Analysis

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Antibodies used in the experiments with mouse cells were obtained from Biolegend unless otherwise stated: anti-CD3 (17A2); anti-CD4 (GK1.5); anti-CD8 (53-6.7); anti-B220 (RA3-6B2); anti-CD25 (PC61.5); anti-CD44 (IM7); anti-CD45 (30-F11); anti-CD62L (MEL-14); anti-CD45.1(A20); anti-CD45.2 (104); anti-NKG2D (CX5); anti-PD-1 (29F.1A12); anti-TIM-3 (RMT3-23); anti-CD200 (OX-90); anti-EpCAM (G8.8); anti-MHC I (36-7-5); anti-MHC II (M5/114.15.2); anti-LAG-3 (C9B7W; Thermo Fisher Scientific); anti-pan NK cells (DX5; Thermo fisher); anti-Foxp3 (FJK-16s; Thermofisher); IL-17 (TC11-18H10.1); anti-TNF-α (TN3-19.12); anti-IFN-γ (XMG1.2); anti-Eomes (Dan11mag; Thermo fisher); anti-TOX (REA473; Miltenyi Biotec), anti-pStat1 (Tyr701) (58D6; Cell Signaling); anti-pStat5 (Tyr694) (C71E5; Cell Signaling); anti-Actin (SCBT). The following mAbs raised against human antigens were all purchased from Biolegend: CD3 (OKT3); CD4 (OKT4); CD8 (SK1); NKG2D (1D11); IL-17 (BL168); IFN-γ (B27). Viable cell populations were gated based on forward and side scatters and by fixable blue (Thermo Fisher Scientific) staining. Samples were collected on an LSR II and analyzed with FlowJo software (Tree Star).

Dai Z., Sezin T., Chang Y., Lee E.Y., Wang E.H, & Christiano A.M. (2022). Induction of T cell exhaustion by JAK1/3 inhibition in the treatment of alopecia areata. Frontiers in Immunology, 13, 955038.

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Cytotoxic Granule Trafficking Assay

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The following antibodies were used against perforin (dG9), lysosome-associated membrane protein (LAMP) 1–Alexa Fluor 488 (H4A3), CD3 (UCHT1), CD56 (HCD56), and NKG2D (1D11; BioLegend, San Diego, Calif); cation-independent mannose 6-phosphate receptor (CI-MPR; 2G11), dynein heavy chain, and Ras-associated binding protein (Rab) 27a (Abcam, Cambridge, United Kingdom); early endosome antigen 1 ([EEA-1] 14/EEA1; BD Biosciences, San Jose, Calif); cathepsin D, Rab7, and Rab9 (Cell Signaling, Danvers, Mass); LAMP1 (H4A3), LAMP2 (H4B4), granzyme B (GB7), Munc13-4, and Rab14 (D-5; Santa Cruz Biotechnology, Dallas, Tex); myosin IIA and actin (AC-15; Sigma, St Louis, Mo); vesicle associated membrane protein 7 (Synaptic Systems, Goettingen, Germany); LAMP2–Alexa Fluor 488 (H4B4) (eBioscience, San Diego, Calif); and perforin (Pf-344, for immunoblotting; Mabtech, Stockholm, Sweden).

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Comprehensive NK Cell Phenotyping

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For surface marker staining, NK cells were washed with PBS and stained with FITC, PE- and PerCP-conjugated mAb for 20 min; the mAb anti-human CD56 (NCAM16.2), CD16 (B73.1), CD158a (HP-3E4), Nkp44 (p44-8), CD8 (SK1) (all from BD Biosciences, San Jose, CA, USA), CD159a (131,411 purchased from R&D Systems, Minneapolis, MN, USA), NKG2D (1D11) and NKp46 (9E2) (all purchased from Biolegend, San Diego, CA, USA) and TRPA1 (Alomone, Israel) were used followed by anti-rabbit PE (R&D) as a secondary antibody. Mouse IgG isotypes were used as controls (BD Biosciences). For acquisition and analysis, the first was performed using an Attune Nxt (Life Technologies, Carlsbad, CA, USA) cytofluorimeter whereas the second was performed using Flow logic software 7.1 (Miltenyi), according to guidelines for the use of flow cytometry and cell sorting in immunological studies [41 (link)].

Scopelliti F., Dimartino V., Cattani C, & Cavani A. (2023). Functional TRPA1 Channels Regulate CD56dimCD16+ NK Cell Cytotoxicity against Tumor Cells. International Journal of Molecular Sciences, 24(19), 14736.

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Multiparameter Analysis of PBMC and Blister NK Cells

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Multiparameter analysis of PBMC and blister NK cell phenotype was performed on an ARIA II (BD Biosciences). PBMCs and blister cells were stained with different combinations of antibodies specific to human CD3(UCHT1;Biolegend), CD8(HIT8a;Biolegend), CD16(3G8;Biolegend), CD45(HI30;Biolegend), CD56(MEM-188;Biolegend), CD62L(DREG-56;Biolegend), CD69(FN50;Biolegend), CD107a(H4A3;Biolegend), NKG2D(1D11;Biolegend), CXCR6(K041E5;Biolegend), T-bet(04–46; BD), and EOMES(WD1928;ThermoFisher). All surface staining was performed for 30 min on ice after prior incubation of cells with Human Fc block and Murine Fc block (BD) for 10 minutes on ice. Following surface staining, cells were fixed and permeabilized using Foxp3 Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (eBiosciences, Thermo Fisher), as per the manufacturers protocol, and stained for the expression of the transcription factors T-bet and Eomes. Fluorescence minus one control stains were performed using PBMC to verify the staining specificity and as a guide for setting markers to delineate positive and negative populations. Gating was set on the live lymphocyte population using forward- and side-scatter profiles to include lymphocytic blasts, followed by single cell gating using forward- and side-scatter heights and widths, before identification of hematopoietic cells using human CD45 staining.

Nikzad R., Angelo L.S., Aviles-Padilla K., Le D.T., Singh V.K., Bimler L., Vukmanovic-Stejic M., Vendrame E., Ranganath T., Simpson L., Haigwood N.L., Blish C.A., Akbar A.N, & Paust S. (2019). Human Natural Killer Cells Mediate Adaptive Immunity to Viral Antigens. Science immunology, 4(35), eaat8116.

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