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Nkg2d

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NKG2D is a type II transmembrane glycoprotein that functions as an activating receptor on natural killer cells, CD8+ T cells, and γδ T cells. It recognizes stress-induced ligands on target cells and triggers cytotoxicity and cytokine production.

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Lab products found in correlation

Topseal a plus, by PerkinElmer (2 mentions) Luma 96 plates, by PerkinElmer (2 mentions) Nkp46, by R&D Systems (2 mentions) Nkp44, by R&D Systems (2 mentions) Nkp30, by R&D Systems (2 mentions) Triton x, by Merck Group (1 mentions) Triton x 100, by Merck Group (1 mentions) Trilux beta scintillation counter, by PerkinElmer (1 mentions) Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Ficoll paque plus, by Cytiva (1 mentions) Cd3 okt3, by Thermo Fisher Scientific (1 mentions) Nkg2a z199, by Beckman Coulter (1 mentions) Mouse igg1 mopc 21, by Merck Group (1 mentions) Goat anti mouse, by BD (1 mentions) Anti cd107a fitc h4a3, by BD (1 mentions) Anti cd3 percp sk7, by BD (1 mentions) Cd244 2b4 c1.7, by Beckman Coulter (1 mentions) Isotype control mouse igg1 mopc 21, by Merck Group (1 mentions) Actin c4, by BD (1 mentions) Anti cd56 pe ncam16.2, by BD (1 mentions)

11 protocols using nkg2d

1

Cytotoxicity Assay for NK Cells

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Chromium (51Cr) release assays were performed as previously described (41 (link), 42 (link)). In brief, target 721.221 cells were labelled with 51Cr (Perkin Elmer) and co-cultured with dilutions of NK cell and % cytotoxicity was determined in reference to a standard spontaneous (standard cell culture media) and maximum (0.5% Triton X, Sigma) release. All assays were co-cultured for 3 hours and resulting 51Cr release was measured using Luma-96 plates (Perkin Elmer), Top-Seal A Plus (Perkin Elmer) and Top Count NXT Microplate Scintillation and Luminescence Counter. Primary NK cell reverse antibody-dependent cytotoxicity assays were completed using antibodies against NKG2D (R&D Systems), 2B4 (C1.7) and CD16 (3G8) and the P815 cell line as previously described (41 (link)).
Wilton K.M, & Billadeau D.D. (2018). Vasodilator Stimulated Phosphoprotein (VASP) Regulates Natural Killer Cell Lytic Granule Convergence. Journal of immunology (Baltimore, Md. : 1950), 201(10), 2899-2909.
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2

Chromium Release Assay for NK Cell Cytotoxicity

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Chromium (51Cr) release assays were performed as previously described (Ham et al., 2013 (link), 2015 (link)). In brief, target 721.221 cells were labeled with 51Cr (Perkin-Elmer) and co-cultured with dilutions of NK cells, and cytotoxicity percent was determined in reference to a standard spontaneous (standard cell culture media) and maximum (0.5% Triton X-100; Sigma-Aldrich) release. All assays were performed for 4 h, and resulting 51Cr release was measured using Luma-96 plates (PerkinElmer), Top-Seal A Plus (PerkinElmer), and Top Count NXT Microplate Scintillation and Luminescence Counter. NK cell reverse antibody-dependent cytotoxicity assays (R-ADCC) were performed using antibodies against NKG2D (R&D Systems), 2B4 (C1.7) and CD16 (3G8), and the P815 cell line, as previously described (Ham et al., 2013 (link)).
Phatarpekar P.V., Overlee B.L., Leehan A., Wilton K.M., Ham H, & Billadeau D.D. (2020). The septin cytoskeleton regulates natural killer cell lytic granule release. The Journal of Cell Biology, 219(11), e202002145.
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3

Docetaxel-Induced PBMC Cytotoxicity Assay

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Breast carcinoma cells were treated or not (controls) with docetaxel (100 nM) for 6, 12 or 72 hours and labeled with 100 μCi 51Cr (Perkin-Elmer, Waltham, MA, USA) for 1 hour at 37°C. After 3 washes with PBS-5% FBS, cells were co-incubated for 4 hours at 37°C with PBMCs isolated by density-gradient separation using Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ, USA) from healthy donors (effector:target ratio 50:1) in 200 μl RPMI 1640 complete medium (Gibco, Waltham, MA, USA) in triplicate 96-well U-bottomed plates in the presence of saturating concentrations of trastuzumab (4 μg/ml). NKG2D (1 μg/ml; R&D Systems) was used in blocking experiments. Radioactivity of the supernatant was measured with a Trilux Beta Scintillation Counter (Perkin-Elmer). Percent specific lysis was calculated as: 100 × (experimental cpm – spontaneous cpm)/(maximum cpm – spontaneous cpm).
Di Modica M., Sfondrini L., Regondi V., Varchetta S., Oliviero B., Mariani G., Bianchi G.V., Generali D., Balsari A., Triulzi T, & Tagliabue E. (2015). Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition. Oncotarget, 7(1), 255-265.
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4

Immobilizing Antibodies for NK Cell Assays

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To immobilize different antibodies in a comparable way to E-plates, we decided to coat the wells first with goat-anti-mouse antibodies. Therefore, E-Plates 16 PET were pre-coated with 50 μl of 5 μg/ml goat-anti-mouse antibody for 3 hours at 37 °C and subsequently washed three times with PBS. In pilot experiments we determined that the subsequent coating with an antibody against NK cell surface receptors gave the strongest signal (Fig. S1). In contrast, binding the antibody first to the NK cells or adding the antibody at the same time as the NK cells without washing was less effective (Fig. S1). Therefore, primary antibody was added to the pre-coated wells at 2 μg/ml in a volume of 50 μl and incubated at 37 °C for 1 hour. Plates were washed three times with PBS and background reading was performed in 100 μl medium.
The following antibodies were used: CD16 (3G8), 2B4 (C1.7), NKp46 (9E2), NKp80 (5D12), CD28 (28.2), DNAM-1 (TX25), CD56 (NCAM) (all from Biolegend), CD3 (OKT3, eBioscience), NKG2D (R&D), NKp30 (p30-15, produced in our laboratory), NKp44 (p44-2, produced in our laboratory), Mouse IgG1 (MOPC-21, Sigma), CD45 (BD Transduction laboratories), NKG2A (Z199, Beckman Coulter), goat anti-mouse (dianova), and CD94 (HP-3D9, BD Bioscience).
Fasbender F, & Watzl C. (2018). Impedance-based analysis of Natural Killer cell stimulation. Scientific Reports, 8, 4938.
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5

Comprehensive Antibody Detection Protocol

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The following antibodies (Ab) were used to detect the indicated proteins: Isotype control mouse IgG1 (MOPC-21; Sigma), NKG2D (149810; R&D Systems), CD244/2B4 (C1.7; Beckman Coulter), actin (C4; BD Biosciences), and p-ERK1/2 (9101), ERK1/2 (9102), pS473-Akt (9271), and FLNa (4762) (all from Cell Signaling). The Abs used for flow cytometry included anti-CD107a-FITC (H4A3), anti-CD56-PE (NCAM16.2), and anti-CD3-PerCP (SK7) (all from BD Biosciences).The fluorochrome-conjugated Ab and reagents used for confocal microscopy were the Vybrant CFDA (CFSE) SE cell tracer kit (V12883), Cell tracker Orange CMTMR (C2927), Alexa Fluor 488 phalloidin (A12379) (all from Invitrogen), Alexa Fluor 647 anti-human perforin (308109; Biolegend), and anti-human FLNa (MAB1678; Chemicon). Secondary anti-mouse and anti-rabbit antibodies conjugated with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology.
Kim N., Yi E., Kwon S.J., Park H.J., Kwon H.J, & Kim H.S. (2022). Filamin A Is Required for NK Cell Cytotoxicity at the Expense of Cytokine Production via Synaptic Filamentous Actin Modulation. Frontiers in Immunology, 12, 792334.
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6

Protein Isolation and Quantification from EVs and Cells

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Total protein was isolated from EVs and cultured cells with a radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific), and protein concentrations were detected by a BCA protein assay kit (Pierce, Rockford, IL, USA). Thirty micrograms of proteins from each sample was separated on an 8% SDS-PAGE gel and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). Membranes were blocked with 2% BSA in TBS containing 0.1% Tween 20 at 37°C for 2 hr and then incubated for 2 hr with either CD63 (Santa Cruz, CA, USA), Calnexin (Cell Signaling Technology), NKG2D (R&D Systems), FasL (R&D Systems), TNF-α (NOVUS, Littleton, CO, USA), IFN-γ (NOVUS), perforin (Invitrogen), tubulin (NOVUS), MHC class I (Abnova, Beijing, China), MHC class II (NOVUS), CD80 (Bio-Rad Laboratories, Hercules, CA, USA), CD86 (R&D Systems), and CD40 (R&D Systems). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG was used as a secondary antibody (diluted 1:5,000 in TBST with 2% BSA and incubated for 1 hr). Bands were scanned using a densitometer (GS-700; Bio-Rad Laboratories), and quantification was performed using Quantity One 4.4.0 software.
Li L., Lu S., Liang X., Cao B., Wang S., Jiang J., Luo H., He S., Lang J, & Zhu G. (2018). γδTDEs: An Efficient Delivery System for miR-138 with Anti-tumoral and Immunostimulatory Roles on Oral Squamous Cell Carcinoma. Molecular Therapy. Nucleic Acids, 14, 101-113.
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7

Phenotypic Profiling of NK Cells

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The following antibodies were used: CD34 (PercpCy5.5 or PE, clone 581), CD56 (PercpCy5.5 or PeCy7, clone B159), CD94 (FITC, clone HP-3D9), CD117 (PeCy7, clone 104D2 or PercpCy5.5, clone YB5.B8), CD161 (FITC,clone DX12), CD14 (ApcCy-7, clone MφP9), CD158a (FITC clone, HP3E4), CD158b (FITC, clone CH-2), NKB1 (FITC, clone DX9) all from BD Biosciences, San Jose, CA. Additional antibodies included NKG2D (PE, clone 149810), NKp30 (PE, clone 210845), NKp44 (PE, clone 253415), and NKp46 (PE, clone 195314) all obtained from R&D Systems, Minneapolis, MN. Intracellular staining for IFN-γ (PE clone 45.B3) was performed using cytofix/cytoperm (BD Biosciences). IFN-γ staining was performed after 16 hours of stimulation with IL-12 (10 ng/mL) and IL-18 (100 ng/mL). Brefeldin A was added for the last 4 hours. Data were analyzed using Flowjo Version 7.6. FACS sorting was performed on either a FACS Vantage or FACS Aria (BD Biosciences).
Weeres M.A., Robien K., Ahn Y.O., Neulen M.L., Bergerson R., Miller J.S, & Verneris M.R. (2014). The Effects of 1,25 Dihydroxyvitamin D3 (1,25(OH)2D3) on In Vitro Human Natural Killer Cell DevelopmentFrom Hematopoietic Stem Cells. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3456-3462.
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8

Characterizing Tumor Cell Immunophenotype

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The harvested tumor from ectopic allograft model were digested into single cells. Tumoral cells and cultured human or mouse HCC cells (5 × 105) were incubated with fluorescent dye-conjugated mAb (MICA, MICB, RAE1, H60, NKG2D, PD-L1) (R&D Systems, Minneapolis, MN, USA) or the isotype controls at 4°C for 1 h. Then, cells were washed three times with PBS. The fluorescence was finally detected with FACS Calibur (BD Biosciences, San Jose, CA, USA) and analyzed with BD CellQuest Pro software.
Liu Y.F., Chiang Y., Hsu F.M., Tsai C.L, & Cheng J.C. (2022). Radiosensitization effect by HDAC inhibition improves NKG2D-dependent natural killer cytotoxicity in hepatocellular carcinoma. Frontiers in Oncology, 12, 1009089.
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9

NKR Ligand Expression Assay

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NKG2D, NKp30, NKp44, and NKp46 Fc chimeras were purchased from R&D Systems (Minneapolis, MN) and added to cytotoxicity assays at 10 μg/ml along with 10 μg/ml donkey anti-human F(ab′)2 fragments (Jackson ImmunoResearch Laboratories, West Grove, PA) to block Ab-dependent cellular cytotoxicity. Expression of NKG2D and NCR ligands was accomplished by staining tumor cells with 1 μg of the aforementioned Fc chimeras and detecting with a PE-conjugated anti-human secondary Ab (Jackson ImmunoResearch Laboratories).
Ames E., Canter R.J., Grossenbacher S.K., Mac S., Chen M., Smith R.C., Hagino T., Perez-Cunningham J., Sckisel G.D., Urayama S., Monjazeb A.M., Fragoso R.C., Sayers T.J, & Murphy W.J. (2015). NK Cells Preferentially Target Tumor Cells with a Cancer Stem Cell Phenotype. Journal of immunology (Baltimore, Md. : 1950), 195(8), 4010-4019.
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10

Redirected Cytotoxicity Assay for NK Cells

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Redirected cytotoxicity assays were performed as described(4 (link)). Briefly, P815 cells were incubated with 5 μg/ml of the indicated combinations of mAbs to CD28H (R&D MAB83162), 2B4 (BioLegend 329502), NKp46 (BD 557847), NKG2D (R&D MAB139), CD2 (BD 555323), DNAM-1 (BD Biosciences 559787), CD16 (BD 555404) and CD56 (BD 555513) for 15 minutes at room temperature. Resting NK cells were added at an E:T ratio of 1:2, mixed and gently centrifuged at 300 rpm for 1 minute. After 2 hours at 37°C, cells were stained with Live/Dead-NIR (Thermo Fisher), anti–CD56-Bv421 (BD 562751) and anti–CD107a-PE (BD 555801) and analyzed by flow cytometry. Target cell lysis assays were either performed using the ToxiLight Non-Destructive Cytotoxicity BioAssay Kit (Lonza) following the manufacturer’s instruction, or through a flow-based assay. Briefly, NK cells were incubated with PKH67-labeled target cells for 6 hours in IMDM medium with 10% FCS at the indicated E:T ratios. Cells were stained with Live/Dead NIR, and the lysis of target cells were determined by flow cytometry. For CD28H blocking, NK cells were pre-incubated with 10 μg/ml CD28H antibody (R&D Systems) for 15 min before mixing with 221.B7H7 cells. KHYG-1 and NKL cells were rested in complete IMDM medium without IL-2 for 1 day, prior to use in cytotoxicity assays.
Zhuang X, & Long E.O. (2019). CD28 homolog is a strong activator of natural killer cells for lysis of B7H7+ tumor cells. Cancer immunology research, 7(6), 939-951.
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