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Dead end kit

Manufactured by Promega
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The Dead-End kit is a laboratory product designed for the detection and quantification of double-strand DNA breaks. It provides a straightforward method to label and analyze the ends of DNA fragments, enabling researchers to assess cellular DNA damage and repair processes.

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Lab products found in correlation

Cleaved caspase 3, by Sartorius (2 mentions) A0398, by Agilent Technologies (2 mentions) Rmr622l, by Biocare Medical (2 mentions) F4 80, by Cell Signaling Technology (2 mentions) Ly 6b 2, by Novus Biologicals (2 mentions) D2sr9, by Novus Biologicals (2 mentions) Neun antibody, by Merck Group (1 mentions) Eclipse 80i fluorescence microscope, by Nikon (1 mentions) Videoconfocal vico system, by Nikon (1 mentions) Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions) Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions) Rabbit on rodent hrp polymer, by Biocare Medical (1 mentions)

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DEAD End TUNEL kit (6 citations) Dead-End Fluorimetric Kit (4 citations)

14 protocols using dead end kit

1

TUNEL and NeuN Double Staining

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TUNEL staining was performed using Promega DeadEnd Kit. Briefly, sections were fixed with 4% paraformaldehyde. After equilibration, sections were incubated with TdT reaction mix for 1hr at 37°C. Stop the reaction with 2xSSC and then mount and analyze. For double staining with NeuN, before TUNEL staining, sections were incubated with NeuN antibody (Milipore) followed by Alexa Fluor 647- tagged secondary antibody.
Shen Y., McMackin M.Z., Shan Y., Raetz A., David S, & Cortopassi G. (2016). Frataxin Deficiency Promotes Excess Microglial DNA Damage and Inflammation that Is Rescued by PJ34. PLoS ONE, 11(3), e0151026.
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2

Fluorometric TUNEL Assay for Cell Death

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Fluorometric TUNEL assay was performed using a DeadEnd Kit (Promega, Madison, WI), in accordance with the manufacturer's protocols.
Goldsmith J.R., Spitofsky N., Zamani A., Hood R., Boggs A., Li X., Li M., Reiner E., Ayyaz A., Etwebi Z., Lu L., Rivera Guzman J., Bou-Dargham M.J., Cathoupolis T., Hakonarson H., Sun H., Wrana J.L., Gonzalez M.V, & Chen Y.H. (2020). TNFAIP8 controls murine intestinal stem cell homeostasis and regeneration by regulating microbiome-induced Akt signaling. Nature Communications, 11, 2591.
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3

Evaluating Apoptosis in Brain Ischemia

Cited 1 time
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To evaluate apoptosis, TUNEL staining was performed on brain sections using a Deadend kit (Promega). Multiple sections from individual brain samples were layered on a glass slide before the TUNEL assay; brain sections were fixed in 4% (vol/vol) paraformaldehyde and permeablized with 0.2% Triton X-100 and proteinase K as per manufacturer’s instructions. After staining, the sections were mounted in hardset Vectashield medium containing DAPI. Additional staining was done with hematoxylin–eosin to locate the boundary of the ischemic penumbra (Borlongan, et al., 2000 (link)). The sections were photographed using a fluorescence microscope equipped with AxioVision software. Four wild type and four Xrcc1+/− brains were analyzed without knowledge of the genotype of the mice. A minimum of four microscopic fields within the ischemic penumbra of each section were photographed. TUNEL+ cells were counted in a double-blinded manner in each of these areas. An average of all of the TUNEL+ cells from these areas counted was taken from each brain section (Khan, et al., 2009 (link)). Data were analyzed by Student’s two-tailed t-test.
Ghosh S., Canugovi C., Yoon J.S., Wilson DM I.I.I., Croteau D.L., Mattson M.P, & Bohr V.A. (2015). Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice. Neurobiology of aging, 36(7), 2319-2330.
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4

Histopathological Analysis of Lungs and Intestines

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Lungs and intestines were fixed in 10% formalin, then processed and embedded in paraffin by standard procedures. Sections (5 μM) were stained with hematoxylin and eosin (H&E) and examined by a pathologist blinded to the experimental groups. For immunohistochemistry, formalin-fixed paraffin-embedded lungs and intestines were cut into 4 μM sections. Cleaved caspase-3 (Essen Bioscience, 4704) and CD45 (BD PharMingen™, 553076) staining was performed according to the manufacturer’s instructions. TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling) staining was performed using the Dead-End kit (Promega, PRG7130) according to the manufacturer’s instructions. The number of Cleaved caspase-3– and TUNEL-positive cells in five high power fields (20x) were counted per mouse.
Karki R., Sharma B.R., Tuladhar S., Williams E.P., Zalduondo L., Samir P., Zheng M., Sundaram B., Banoth B., Malireddi R.K., Schreiner P., Neale G., Vogel P., Webby R., Jonsson C.B, & Kanneganti T.D. (2020). Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes. Cell, 184(1), 149-168.e17.
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5

Apoptosis Detection via TUNEL Assay

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For apoptosis detection, the Terminal transferase-mediated dUTP-biotin Nick End-Labeling (TUNEL) assay was conducted, using the DeadEnd kit (Promega, Madison, WI, USA). Nuclei were stained with Hoechst 33258. Cells undergoing apoptotic cell death were imaged using an Eclipse 80i Nikon Fluorescence Microscope equipped with a VideoConfocal (ViCo) system (Nikon Instruments, Amsterdam, The Netherlands). Apoptosis was expressed as percentage of TUNEL-positive nuclei cells over total cells (at least 500 cells for each coverslip).
Matteucci A., Varano M., Gaddini L., Mallozzi C., Villa M., Pricci F, & Malchiodi-Albedi F. (2014). Neuroprotective Effects of Citicoline in in Vitro Models of Retinal Neurodegeneration. International Journal of Molecular Sciences, 15(4), 6286-6297.
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6

Histological Analysis of Formalin-Fixed Liver

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Formalin-preserved livers were processed and embedded in paraffin according to standard procedures. Sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined by a pathologist blinded to the experimental groups. For immunohistochemistry, formalin-fixed paraffin-embedded livers were cut into 4 μm sections. To stain for granulocytes, we used an anti-MPO antibody (1:500 dilution for 30 min, A0398, Dako) followed by rabbit on rodent polymer-HRP (RMR622L, BioCare Medical) for 30 min. TUNEL staining was performed using the Dead End kit (#PRG7130) according to the manufacturer’s instructions (Promega).
Man S.M., Karki R., Malireddi R.K., Neale G., Vogel P., Yamamoto M., Lamkanfi M, & Kanneganti T.D. (2015). The transcription factor IRF1 and guanylate-binding proteins target AIM2 inflammasome activation by Francisella infection. Nature immunology, 16(5), 467-475.
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7

Evaluating Cell Viability and Apoptosis

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Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; the MTT assay has been widely used to assess cell viability and is based on the ability of viable cells to reduce MTT, giving rise to an insoluble purple formazan salt. Briefly, the cultures were incubated for 20 minutes at 37 °C with 0.5 mg/ml MTT in Hank’s balanced salt solution (Life Technologies). The reaction product was dissolved in dimethyl sulphoxide. The spectral photometric absorbance of the samples was determined at a wavelength of 540 nm. The amount of MTT conversion was evaluated as a percentage of the absorbance measured in treated cells relative to the absorbance of control cells. After fixation in 4% paraformaldehyde in PBS, 0.12 M in sucrose, apoptosis was evaluated in mixed hippocampal cultures by the terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay (DeadEnd kit, Promega, Madison, WI).
Belfiore M., Cariati I., Matteucci A., Gaddini L., Macchia G., Fioravanti R., Frank C., Tancredi V., D’Arcangelo G, & Diociaiuti M. (2019). Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca2+-influx, LTP impairment and neurotoxicity. Scientific Reports, 9, 5144.
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8

Histological Analysis of Murine and Human Lungs

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For murine samples, lungs were fixed in 10% formalin, then processed and embedded in paraffin by standard procedures. Sections (5 μM) were stained with hematoxylin and eosin (H/E) and examined by a pathologist blinded to the experimental groups. For immunohistochemistry, formalin-fixed paraffin-embedded lungs were cut into 4 μM sections. F4/80 (Cell Signaling, D2SR9) and Ly-6B.2 (Novus, NBP2-13077) staining was performed according to the manufacturer’s instructions. TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling) staining was performed using the Dead-End kit (Promega, PRG7130) according to the manufacturer’s instructions.
For human lung samples, de-identified autopsy lung samples were provided by the Asian Institute of Gastroenterology as paraffin embedded tissue blocks. Sectioning (4 μm) and immunohistochemistry were performed by HistoWiz with hematoxylin and eosin, the rabbit polyclonal anti–SARS-CoV-2 Nucleocapsid (N) protein antibody (GeneTex; GTX635686), and TUNEL (Promega) staining following standard protocols.
Karki R., Lee S., Mall R., Pandian N., Wang Y., Sharma B.R., Malireddi R.S., Yang D., Trifkovic S., Steele J.A., Connelly J.P., Vishwanath G., Sasikala M., Reddy D.N., Vogel P., Pruett-Miller S.M., Webby R., Jonsson C.B, & Kanneganti T.D. (2022). ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection. Science Immunology.
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9

Immunochemical Analysis of Liver Tissues

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Liver tissues were fixed in 10% formalin and embedded in paraffin for immunochemical staining. 4-µm–thick liver sections were stained with anti–cleaved caspase 3 or anti-myeloperoxidase (MPO) followed by incubation for 30 min with Rabbit-on-Rodent HRP polymer (RMR622; BioCare Medical). TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling) staining was performed with the Dead End kit (G7130) according to the manufacturer’s instructions (Promega). Liver tissues were homogenized in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors for immunoblot analysis. Protein concentrations were determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific) according to manufacturer’s instructions.
Qi X., Man S.M., Malireddi R.K., Karki R., Lupfer C., Gurung P., Neale G., Guy C.S., Lamkanfi M, & Kanneganti T.D. (2016). Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection. The Journal of Experimental Medicine, 213(10), 2081-2097.
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10

Histological Analysis of Formalin-Fixed Liver

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Formalin-preserved livers were processed and embedded in paraffin according to standard procedures. Sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined by a pathologist blinded to the experimental groups. For immunohistochemistry, formalin-fixed paraffin-embedded livers were cut into 4 μm sections. To stain for granulocytes, we used an anti-MPO antibody (1:500 dilution for 30 min, A0398, Dako) followed by rabbit on rodent polymer-HRP (RMR622L, BioCare Medical) for 30 min. TUNEL staining was performed using the Dead End kit (#PRG7130) according to the manufacturer’s instructions (Promega).
Man S.M., Karki R., Malireddi R.K., Neale G., Vogel P., Yamamoto M., Lamkanfi M, & Kanneganti T.D. (2015). The transcription factor IRF1 and guanylate-binding proteins target AIM2 inflammasome activation by Francisella infection. Nature immunology, 16(5), 467-475.
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