TUNEL (terminal uridine nick-end labeling) assays were performed to measure the apoptosis by using the Promega DeadEnd™ Fluorometric TUNEL assay system. Cells were cultured on coverslips for 24 h and then treated with hydrogen peroxide solution (H2O2) in the presence or absence of NAC (N-acetylcysteine), delphinidin chloride, chloroquine, and rapamycin. Cells were fixed with 4% (w/v) paraformaldehyde for 30 min and permeabilized with PBS containing 0.1% Triton X-100 for 20 min at room temperature. Cells were blocked with 5% horse serum in PBS for 1 h, and then TUNEL assays were performed to measure the apoptosis accordance with the manufacturer’s instruction of the Promega DeadEnd™ Fluorometric TUNEL assay system for 1 h. After washing with PBS, cells the glass coverslips were mounted onto glass slides using a mounting medium containing DAPI. Slides were analyzed with a florescence microscope (BX51-DSU; Olympus, Tokyo, Japan). More than 1500 nuclei were counted per field. The experiment was repeated three times.
Deadend fluorometric tunel system assay
Manufactured by Promega
Sourced in United States
The DeadEnd Fluorometric TUNEL System assay is a laboratory tool designed to detect and quantify apoptosis, a form of programmed cell death, in cells. The assay utilizes fluorescein-12-dUTP to label the free 3'-OH DNA ends generated during apoptosis, allowing for the direct detection of apoptotic cells.
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Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Axio imager z1, by Zeiss (2 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Rabbit anti glucagon, by Cell Signaling Technology (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Guinea pig anti insulin, by Agilent Technologies (1 mentions)
96 well flat bottom plate, by Corning (1 mentions)
Bx51 dsu, by Olympus (1 mentions)
9 protocols using deadend fluorometric tunel system assay
1
Quantifying Apoptosis via TUNEL Assay
2
TUNEL Assay for Apoptosis Detection
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TUNEL was performed with the DeadEnd™ Fluorometric TUNEL System Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Untreated cells were used as negative control. A positive control was also used but it is not reported in the Fig. 5 . At the end of the treatment, hCFs were fixed with 4% paraformaldehyde solution for 25 minutes at 4 °C, permeabilized with 0.2% Triton X-100 solution in PBS for 5 minutes and finally incubated with 50 µl TdT reaction mix for 60 minutes at 37 °C in a humidified chamber. At the end of incubation, the reaction was blocked with a stop solution and the nuclei were counterstained with Hoechst 33258 (SIGMA-ALDRICH, St. Louis, MO, USA). Slides were mounted with Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA, USA) and TUNEL-positive cells and the total cell number per view were counted with a Zeiss Axio Observer Z1 microscope (Carl Zeiss, Milan, Italy) equipped with the Apotome system.
3
TUNEL Assay for Detecting Apoptosis
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This method is described in detail by Resnick-Silverman.87 (link) The TUNEL assay is performed on 10μM tissue sections. Following dissection, whole organs are transferred into wells containing 4% Paraformaldehyde in PBS overnight at 4°C. The fixation solution is replaced with 30% sucrose and returned to 4°C. After 24h the organs are placed in molds on dry ice and embedded in OCT compound. Once hardened, the molds can be stored at −80°C until they are sectioned. In preparation for the assay, the sectioned tissues are rehydrated with PBS (pH 7.4) followed by permeabilization with 0.2% Triton X-100 in PBS. The Promega DeadEnd Fluorometric TUNEL System assay is used for the detection of apoptotic cells. The 3′ ends of the damaged DNA can be detected by using the recombinant Tdt enzyme to incorporate fluorescein-12-dUTP at the 3′ OH ends and is provided by the manufacturer. The steps are followed as per the kit instructions.
After termination of the reaction, Vectastain (an antifade fluorescence mounting media) is applied to the section before pressing down the coverslip. The slides are stored overnight at −20°C and analyzed the following day on a Zeiss Axio Imager.Z1 with Fluorescence and Phase Contrast.
After termination of the reaction, Vectastain (an antifade fluorescence mounting media) is applied to the section before pressing down the coverslip. The slides are stored overnight at −20°C and analyzed the following day on a Zeiss Axio Imager.Z1 with Fluorescence and Phase Contrast.
4
Viability and Morphology of Pancreatic Islets
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Cell viability was detected by calcein AM (live cells) and propidium iodide (dead cells) dye staining according to the manufacturer´s instruction (Thermo Fisher Scientific). Samples were analyzed under a fluorescent microscope (n > 50 cluster) and by flow cytometry at day 7 after isolation. To determine the cluster architecture, NPICCs and REPIs were embedded in Epredia™ HistoGel™ Specimen Processing Gel (Thermo Fisher Scientific) and stained for insulin (guinea pig anti-insulin, 1:400, Agilent-Dako, Frankfurt, Germany) and glucagon (rabbit anti-glucagon, 1:100, Cell Signaling, Frankfurt, Germany) followed by incubation with FITC-labelled anti-rabbit IgG and Cy3-labeled anti-guinea pig IgG (Thermo Fisher Scientific). DAPI was used to counterstain cell nuclei. Recovery rate was determined by calculation of the ratio IEQ of REPIs to IEQ of native NPICCs. Apoptotic cells were analyzed on day 7 by TUNEL staining of NPICCs and REPIs using the DeadEnd Fluorometric TUNEL System assay according to the manufacturer´s instructions (Promega, Madison, WI, United States).
5
TUNEL Assay for DNA Fragmentation
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DNA fragmentation was detected by TUNEL, using the DeadEnd™ Fluorometric TUNEL System assay (Promega Corporation, Madison, WI, USA). Cells (5×103) were plated into 96-well flat bottom plates (Corning Inc., Acton, MA, USA) and allowed to attach overnight prior to treatment with IL-6 (25 ng/ml), IL-6 + AG490 (50 μM), IL-6 + S3I-201 (300 μM), IL-6 + AG490 + TRAIL (25 ng/ml optimal effective dose) and IL-6 + S3I-201 + TRAIL for 24 h in fresh complete medium. The assay was performed as previously described by Kristjansdottir et al (35 (link)). Experiments were performed in triplicate.
6
Apoptosis Detection in Testis Tissue
7
TUNEL Assay for Detecting Apoptosis
8
Apoptosis Evaluation by TUNEL Assay
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Apoptosis was measured by detecting
DNA fragmentation using the DeadEnd Fluorometric TUNEL Assay System
(Promega Corporation, Fitchburg, WI) in accordance with the manufacturer’s
instructions. HeLa, SiHa, PC9, 16HBE, and A549 cells were inoculated
into six-well plates (3 × 104 cells/well). The cells
were treated for 96 h with 5 μM FA-GFLG-MMC in DMEM with a maximum
of 0.1% DMSO. Nuclei were stained with 4′,6-diamidino-2-phenylindole
(DAPI). The cells were washed with PBS three times and immediately
examined under a fluorescent microscope using a standard fluorescent
filter set to view the green fluorescence of fluorescein-12-dUTP at
520 ± 20 nm. DAPI (blue) fluorescence was detected by excitation
using a UV filter.
DNA fragmentation using the DeadEnd Fluorometric TUNEL Assay System
(Promega Corporation, Fitchburg, WI) in accordance with the manufacturer’s
instructions. HeLa, SiHa, PC9, 16HBE, and A549 cells were inoculated
into six-well plates (3 × 104 cells/well). The cells
were treated for 96 h with 5 μM FA-GFLG-MMC in DMEM with a maximum
of 0.1% DMSO. Nuclei were stained with 4′,6-diamidino-2-phenylindole
(DAPI). The cells were washed with PBS three times and immediately
examined under a fluorescent microscope using a standard fluorescent
filter set to view the green fluorescence of fluorescein-12-dUTP at
520 ± 20 nm. DAPI (blue) fluorescence was detected by excitation
using a UV filter.
9
Apoptosis Measurement in Arsenite-Treated HeLa Cells
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Stably transfected HeLa cells expressing pcDNA3-Myc empty vector or pcDNA-Myc-MRS (wild type, SA and SD) were treated with 4 µM sodium arsenite for 72 h and apoptosis was measured using Dead End Fluorometric TUNEL assay system (Promega,) according to the manufacturer's manual. ERK inhibitor was added 1 h before arsenite treatment when required.
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