Tunel kit

Manufactured by R&D Systems

Sourced in United States

The TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) kit is a laboratory tool used to detect and quantify apoptosis, a type of programmed cell death, in various cell and tissue samples. The kit utilizes an enzymatic reaction to label the free 3'-hydroxyl ends of fragmented DNA, which is a hallmark of apoptosis. The labeled DNA can then be visualized and analyzed using various detection methods, such as fluorescence microscopy or flow cytometry. The TUNEL kit provides a standardized and reliable method for researchers to study the mechanisms and pathways involved in apoptosis within their experimental systems.

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9 protocols using tunel kit

Analyzing Cardiac Remodeling Post-AMI

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The animals were sacrificed 3 days or 3 weeks after AMI. The tissues were fixed in formalin and embedded in paraffin blocks according to established protocols. The fixed hearts were serially cut at 8 µm from the apex to the level just below the coronary artery ligation site. After antigen retrieval, the specimens were incubated with 1% normal blocking serum in PBS for 60 min to suppress the nonspecific binding of IgG. The slides were then incubated for 30-60 min with each mouse antibody or fluorescent reagent.
Three days after the AMI, the infarcted tissue was stained with EdU (Invitrogen) to identify the live MSCs, and the TUNEL kit (R&D Systems) was used to identify the nuclei of the apoptotic cardiac myocytes in the infarct border zone. Three weeks after the AMI, the infarct border zone was stained with Texas Red-X-conjugated wheat germ agglutinin (WGA, Invitrogen) and FITC-conjugated CD31 (BD Biosciences) to measure the vessel density. The tissue was further stained with anti-myosin heavy chain eFluor 660 (eBioscience) and 4, 6-diamidino-2-phenylindole (DAPI, Roche) to measure the cardiac myosin-positive area in the infarct zone with Image Pro Plus software. Fluorescence microscopy (Olympus BX61) or a confocal laser scanning (CLS) microscopy system (Thorlabs, Inc.) was used as necessary to obtain images of the immunostaining results.

Lu W., Xie Z., Tang Y., Bai L., Yao Y., Fu C, & Ma G. (2015). Photoluminescent Mesoporous Silicon Nanoparticles with siCCR2 Improve the Effects of Mesenchymal Stromal Cell Transplantation after Acute Myocardial Infarction. Theranostics, 5(10), 1068-1082.

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Apoptosis Quantification via TUNEL Staining

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The cell suspension was seeded into confocal culture dishes and placed in 50 mL/L CO2 cell incubators at 37 °C for routine culture. When the cell confluence was above 80%, the TUNEL staining was performed according to the instructions of TUNEL kit (R&D system, Minneapolis, MN, USA). Finally, the nuclei were stained with 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Wuhan Google Biotech Co., Ltd., Wuhan, China) for 10 min. The apoptotic cells were observed under a confocal microscope (Olympus) to calculate the TUNEL-positive rate.

Wang S., Liu B., Li F., Tang Z., Gu X, & Yuan X. (2024). Identification of the novel biomarkers involved in the mitochondrial metabolism-related reactive oxygen species and their role in lung cancer T-cell exhaustion and immunotherapy. Heliyon, 10(5), e27022.

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TUNEL Staining and Netrin-1 Immunodetection

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Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and netrin-1 immunochemistry were performed by the Anipath core facility (Inserm, Lyon, France), using the R&D systems TUNEL kit and an antibody directed against netrin-1 (MAB1109; R&D Systems) (Supplementary Table 5).

Lahlali T., Plissonnier M.L., Romero-López C., Michelet M., Ducarouge B., Berzal-Herranz A., Zoulim F., Mehlen P, & Parent R. (2016). Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein Response. Cellular and Molecular Gastroenterology and Hepatology, 2(3), 281-301.e9.

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Comprehensive Biochemical Analyses of Tissue Samples

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Creatinine (Cr) assay kit (R&D Systems, Minneapolis, MN, USA); blood urea nitrogen (BUN) assay kit (R&D Systems); xanthine oxidase assay kit used to determine the activity of superoxide dismutase (SOD; R&D Systems); TUNEL kit (R&D Systems); dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA), TRIzol kit (Invitrogen, Carlsbad, CA, USA), reverse transcription kit (Invitrogen); rabbit anti-Bax-beta, rabbit anti-Bax and rabbit anti-GAPDH primary antibodies, and horseradish peroxidase labeled anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA, USA); ECL luminescent substrate (Invitrogen); color developing powder (Invitrogen); pipettes (Eppendorf, Hamburg, Germany); PCR instrument (Applied Biosystems, Inc., Foster City, CA, USA); UV imaging system (Biometra GmbH, Göttingen, Germany); electronic balance (Sartorious BP121S; Sartorius AG, Goettingen, Germany); −80°C refrigerator (Thermo Fisher Scientific, Schwerte, Germany); low temperature centrifuge (Thermo Fisher Scientific) and frozen slicers (Leica Microsystems, Wetzlar, Germany). The sources of other related instruments and reagents are described in the relevant section.

Shen S., Zhou J., Meng S., Wu J., Ma J., Zhu C., Deng G, & Liu D. (2017). The protective effects of ischemic preconditioning on rats with renal ischemia-reperfusion injury and the effects on the expression of Bcl-2 and Bax. Experimental and Therapeutic Medicine, 14(5), 4077-4082.

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LPS-Induced Oxidative Stress and Inflammatory Response

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LPS from Escherichia coli 055:B5 was purchased from Sigma-Aldrich (USA). Commercial enzyme-linked immunosorbent assay (ELISA) kits to assess the levels of the cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit were obtained from R&D Systems (USA). Radioimmunoprecipitation assay (RIPA) lysis buffer and superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) assay kits were purchased from Beyotime Co. Ltd. (China). Acetonitrile and methanol (high-performance liquid chromatography [HPLC] grade) were obtained from Thermo Fisher Scientific (USA). Ultra-pure distilled water was prepared using a Milli-Q purification system (Millipore Corp., USA). All other reagents (analytical grade) were obtained from Sigma-Aldrich (USA).

Gao H., Yang T., Chen X, & Song Y. (2021). Changes of Lipopolysaccharide-Induced Acute Kidney and Liver Injuries in Rats Based on Metabolomics Analysis. Journal of Inflammation Research, 14, 1807-1825.

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CO2 Euthanasia and Tissue Histology

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Animals were sacrificed by CO₂ inhalation. Sections were immediately placed in 10% buffered formalin until paraffin embedding and sectioning (done by the Rodent Histopathology core service at Harvard Medical School). Hematoxylin/Eosin staining was performed by the histopathology core. Immunohistochemical studies were performed on skin specimens using formalin-fixed, paraffin-embedded tissue. Tissues known to express the antigen of interest were used as positive controls, whereas removal of the primary antibodies in the test tissues was used as negative controls. TUNEL kit was purchased from R & D and applied according to the manufacturer's instructions.

Li X., Miao X., Wang H., Xu Z, & Li B. (2015). The tissue dependent interactions between p53 and Bcl-2 in vivo. Oncotarget, 6(34), 35699-35709.

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Apoptosis Quantification in Rat Tissues

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Rats were euthanized, and paraffin sections were prepared as above method. TUNEL assay was conducted using a TUNEL kit (R&D Systems, Minnesota, USA), according to the manufacturer’s protocols. Five fields of vision were randomly selected under an optical microscope (Leica, Wetzlar, Germany), positive brown cells and total cells were counted, and the apoptosis rate was calculated.

Huang R., Shu J., Dai X., Liu Y., Yu F, & Shi G. (2020). The protective effect of polyphyllin I on myocardial ischemia/reperfusion injury in rats. Annals of Translational Medicine, 8(10), 644.

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Histological Analysis of Post-AMI Cardiac Tissue

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The animals were sacrificed on specific days following AMI. The tissues were fixed in formalin and embedded in paraffin blocks according to established protocols. The fixed hearts were serially cut 8 μm from the apex to the level just below the coronary artery ligation site. After antigen retrieval, the specimens were incubated with 1% normal blocking serum in PBS for 60 min to suppress the nonspecific binding of IgG. The slides were then incubated for 30-60 min with each mouse antibody or fluorescent reagent. A TUNEL kit (R&D Systems) was used to identify the nuclei of the apoptotic cardiac myocytes in the infarct border zone on days 3 and 7. CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1/CD31, BD Biosciences) was used for immunohistochemistry to measure vessel density at day 3 and day 21. Microscopy (Olympus BX61) was used as necessary to obtain images of the immunostaining results.

Lu W., Ma G., Sheng Z., Wang Q., Chen L., Qi J., Shi R., Ji J., Ji Z, & Dai Q. (2020). MSCs Contribute to the Conversion of Ly6Chigh Monocytes into Ly6Clow Subsets under AMI. Stem Cells International, 2020, 2460158.

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Quantifying Apoptosis via TUNEL Assay

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The cell suspension was seeded into confocal culture dishes and placed in 50 mL/L CO₂ cell incubators at 37° C for routine culture. When the cell confluence was above 80%, the TUNEL staining was performed according to the instructions of TUNEL kit (R&D system, Minneapolis, MN, USA). Finally, the nuclei were stained with 10 μg/mL 4',6-diamidino-2-phenylindole (DAPI; Wuhan Google Biotech Co., Ltd., Wuhan, China) for 10 min. The apoptotic cells were observed under a confocal microscope (Olympus) to calculate the TUNEL-positive rate.

Diao L, & Zhang Q. (2021). Transfer of lncRNA UCA1 by hUCMSCs-derived exosomes protects against hypoxia/reoxygenation injury through impairing miR-143-targeted degradation of Bcl-2. Aging (Albany NY), 13(4), 5967-5985.

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