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Deadend colorimetric apoptosis detection system

Manufactured by Promega
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The DeadEnd Colorimetric Apoptosis Detection System is a laboratory product designed to detect and quantify apoptosis, a form of programmed cell death. The system utilizes the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay technique to label and detect DNA fragmentation, a hallmark of apoptosis. The assay provides a simple, convenient, and reliable method for the detection and quantification of apoptotic cells in various sample types.

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Lab products found in correlation

Silanized slides, by Agilent Technologies (1 mentions) Hematoxylin, by Thermo Fisher Scientific (1 mentions) Envision system hrp, by Agilent Technologies (1 mentions) Ki 67 antibody, by Agilent Technologies (1 mentions) 3 3 diaminobenzidine dab chromogen, by Vector Laboratories (1 mentions) Permount, by Thermo Fisher Scientific (1 mentions) Formalin, by Merck Group (1 mentions) Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions) Bx40 light microscope, by Olympus (1 mentions)

22 protocols using deadend colorimetric apoptosis detection system

1

TUNEL Assay for Apoptosis Detection

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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using the DeadEnd Colorimetric Apoptosis Detection System (Promega) as previously described.35 (link),36 (link) Briefly, PAECs from each experimental group (control, LPA, and shunt) were treated with tumor necrosis factor (TNF)-α 50 ng/mL. After TNF-α treatment, cells were appropriately washed and fixed, then incubated with terminal deoxynucleotidyl transferase and reaction mix including fluorescein-12-dUTP for 1 h at 37℃. Cells were then again washed and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (5 µM) for 15 min at room temperature. DAPI, a nuclear stain, was used normalize for total cell number. Comparisons were made between baseline conditions and with the addition of either ET-1 (1 µM/mL) or BQ788 (an ETB receptor antagonist, 1 µM/mL).
Zhu T., Chiacchia S., Kameny R.J., Garcia De Herreros A., Gong W., Raff G.W., Boehme J.B., Maltepe E., Lasheras J.C., Black S.M., Datar S.A, & Fineman J.R. (2020). Mechanical forces alter endothelin-1 signaling: comparative ovine models of congenital heart disease. Pulmonary Circulation, 10(2), 2045894020922118.
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2

TUNEL Assay for Apoptosis Detection

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Apoptotic cells in the 6 μm thick frozen sections were detected by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) with the DeadEnd colorimetric Apoptosis Detection System (Promega), according to the manufacturer's protocol. The apoptotic index was defined as the percentage of immunopositive cells in 10 high-power fields (×400).
Matsushima H., Mori T., Ito F., Yamamoto T., Akiyama M., Kokabu T., Yoriki K., Umemura S., Akashi K, & Kitawaki J. (2016). Anti-tumor effect of estrogen-related receptor alpha knockdown on uterine endometrial cancer. Oncotarget, 7(23), 34131-34148.
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3

In Situ Apoptosis Quantification in Tumor Samples

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Apoptosis was determined by TUNEL, using the Fluorometric in situ Cell Death Detection kit (Promega), according to manufacturer's instructions. Ten high-magnification fields were counted and the proportion of apoptotic (green) cells was determined, as the fraction of total cells. For assessment of apoptosis in vivo after Andes-1537 treatment, tumors from ASO-C- or Andes 1537-treated mice were surgical resected, fixed immediately in neutral-buffered formalin (10%) and paraffin-embedded. Afterward, 5 μm-thick serial paraffin sections were collected on silanized slides (DAKO) and hydrated as described before11 (link),15 (link). The TUNEL procedure was performed using DeadEnd™ Colorimetric Apoptosis Detection System (Promega), according to manufacturer's instruction. As positive control, DNAse I-treated muscle and liver sections were included. Control tumor sections were counter-stained with contrast blue (KPL).
Borgna V., Lobos-González L., Guevara F., Landerer E., Bendek M., Ávila R., Silva V., Villota C., Araya M., Rivas A., López C., Socias T., Castillo J., Alarcón L., Burzio L.O., Burzio V.A, & Villegas J. (2020). Targeting antisense mitochondrial noncoding RNAs induces bladder cancer cell death and inhibition of tumor growth through reduction of survival and invasion factors. Journal of Cancer, 11(7), 1780-1791.
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4

Quantifying Tumor Cell Apoptosis

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Mice from each group were sacrificed on day 8. The tumor was fixed in 10% neutral buffered formalin and processed for histopathological and immunohistochemical staining as well as Western blot assay. After fixation, tumor tissues were embedded in paraffin blocks and sectioned (5 μm). Tumor cells were detected in representative stained sections. The expressions of phospho-Akt and phospho-mTOR were evaluated after immunohistochemical staining using specific antibodies. DNA fragmentation was assessed using the DeadEnd Colorimetric Apoptosis Detection System (Promega, Madison, WI, USA). Tissue sections were deparaffinized by immersing slides in fresh xylene, dehydrated in a graded series of ethanols, and subjected to the terminal deoxynucleotidyltransferase–mediated dUTP-biotin nick-end labeling (TUNEL) assay following instructions of the manufacturer. Nucleotide incorporation was detected by treatment with horseradish peroxidase–conjugated streptavidin and enzyme substrate.
Liu W.L., Gao M., Tzen K.Y., Tsai C.L., Hsu F.M., Cheng A.L, & Cheng J.C. (2014). Targeting Phosphatidylinositide3-Kinase/Akt pathway by BKM120 for radiosensitization in hepatocellular carcinoma. Oncotarget, 5(11), 3662-3672.
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5

Immunohistochemical Analysis of Tissue Sections

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Paraffin-embedded blocks were cut into 5-μm sections. Slides were processed as follows: dewaxed in xylene followed by rehydration in a standard alcohol series. Antigen retrieval was performed by pressure cooking for 20 minutes in citrate buffer (pH 6.0), followed by blocking of endogenous peroxidase activity in 3% H2O2. The primary antibodies were added and incubated overnight at 4°C. Antibodies were detected using a secondary HRP–labeled mouse or rabbit antibody detection system (Dako EnVision+ System-HRP catalog No. k4401, catalog No. k4403) followed by addition of 3,3′-diaminobenzidine (DAB) chromogen (Vector Laboratories) for visualization. Sections were counterstained with hematoxylin (Thermo Fisher Scientific Inc.) and slides were dehydrated sequentially in 70%, 80%, and 100% ethanol, followed by xylene. Slides were coverslipped and mounted in Permount (Thermo Fisher Scientific Inc.). Ki67 antibody (Dako, catalog No. F7268) was used at a 1:100 dilution. HK2 rabbit mAb (2876, Cell Signaling Technology) was used at a 1:300 dilution. TUNEL assay was performed using the DeadEnd Colorimetric Apoptosis Detection System (Promega). All images were captured on an Olympus IX73 fluorescent microscope system and analyzed using CellSens Dimension software.
Agnihotri S., Mansouri S., Burrell K., Li M., Mamatjan Y., Liu J., Nejad R., Kumar S., Jalali S., Singh S.K., Vartanian A., Chen E.X., Karimi S., Singh O., Bunda S., Mansouri A., Aldape K.D, & Zadeh G. (2018). Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(2), 844-855.
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6

Apoptosis Detection in Propofol-Treated Cells

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To detect apoptotic cells, the DeadEnd™ Colorimetric Apoptosis Detection System from Promega (Madison, WI) was used. The PANC-1 cells were transfected with LOC285194 siRNA or miR-34a inhibitor or E-cadherin siRNA or its controls for 24 h. Then, the cells were plated at a density of 1 × 106 cells/well in 6-well plates and treated with 20 μg/ml of propofol for 72 h. Cell apoptosis was detected as the manufacture’s instruction. The total number of cells was counted using DAPI staining, and the average ratio of TUNEL-positive cells was calculated.
Wang H., Jiao H., Jiang Z, & Chen R. (2020). Propofol inhibits migration and induces apoptosis of pancreatic cancer PANC-1 cells through miR-34a-mediated E-cadherin and LOC285194 signals. Bioengineered, 11(1), 510-521.
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7

Osteoclast Apoptosis Assay with GlcN

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At the end of osteoclastogenic induction, mature hOCs were treated with GlcN (100 and 200 µg/ml) for 72 h. The cells were then rinsed twice with PBS and fixed for 25 min in 4% PFA at room temperature. Apoptotic cells were detected using a DeadEnd Colorimetric Apoptosis Detection system (Promega Corporation) according to the manufacturer's instructions. Moreover, all cells were subjected to hematoxylin staining to reveal nuclei. The cells were mounted in glycerol/PBS (9:1) and observed under a Leica microscope (magnification, ×20; Leica Microsystems GmbH). The apoptotic rate was calculated as the percentage of apoptotic nuclei (dark brown nuclei) compared with the total number of nuclei of osteoclasts, evaluated in triplicate from each experimental sample (10 randomly selected optical fields/sample).
Lambertini E., Penolazzi L., Pandolfi A., Mandatori D., Sollazzo V, & Piva R. (2021). Human osteoclasts/osteoblasts 3D dynamic co-culture system to study the beneficial effects of glucosamine on bone microenvironment. International Journal of Molecular Medicine, 47(4), 57.
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8

Apoptosis Detection in Kidney Tissue

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Apoptosis detection in kidney tissue sections was performed with a DeadEnd™ Colorimetric Apoptosis Detection System (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer’s instructions. TUNEL-positive apoptotic cells were observed under light microscopy.
Li Y., Yan M., Yang J., Raman I., Du Y., Min S., Fang X., Mohan C, & Li Q.Z. (2014). Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation. Stem Cell Research & Therapy, 5(1), 19.
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9

Quantitative TUNEL Assay for Apoptosis

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To assess the percentage of apoptotic cells in CCA nude mouse tissues, an in situ terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using the DeadEnd™ Colorimetric Apoptosis Detection System (Promega, WI, USA) according to the manufacturer’s instructions. TUNEL-positive cells were quantified in at least five x400 power fields of randomly selected tissue sections.
Dokduang H., Jamnongkarn W., Promraksa B., Suksawat M., Padthaisong S., Thanee M., Phetcharaburanin J., Namwat N., Sangkhamanon S., Titapun A., Khuntikeo N., Klanrit P, & Loilome W. (2020). In vitro and in vivo Anti-Tumor Effects of Pan-HER Inhibitor Varlitinib on Cholangiocarcinoma Cell Lines. Drug Design, Development and Therapy, 14, 2319-2334.
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10

Histological and Apoptosis Analysis of Tumor Tissues

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The tumor tissues were fixed with formalin (Sigma-Aldrich) at 4 °C for 24 h, and soaked with 100% alcohol for 5 min. Specimens were embedded with paraffin after incubation with xylene. Finally, the specimens were sectioned into 4 µm slices. The specimens were stained with HE (Sigma-Aldrich) for 15 min at room temperature, and the stained specimens were visualized under a microscope. In addition, the specimen slices were stained by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay with the DeadEnd™ Colorimetric Apoptosis Detection System (Promega, Madison, WI, USA) referring to the manufacturer’s instructions. Briefly, the slides were incubated by equilibration buffer for 10 min, and treated with proteinase K for 10 min. The samples were washed with PBS and treated with TdT enzyme at 37 °C for 1 h. The slides were then incubated with horseradish peroxidase-labeled streptavidin, and detected with diaminobenzidine. Finally, the images of the specimens were examined under a microscope.
Kung F.P., Lim Y.P., Chao W.Y., Zhang Y.S., Yu H.I., Tai T.S., Lu C.H., Chen S.H., Li Y.Z., Zhao P.W., Yen Y.P, & Lee Y.R. (2021). Piperlongumine, a Potent Anticancer Phytotherapeutic, Induces Cell Cycle Arrest and Apoptosis In Vitro and In Vivo through the ROS/Akt Pathway in Human Thyroid Cancer Cells. Cancers, 13(17), 4266.
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