Terminal Transferase-Mediated dUTP Nick End-Labeling—TUNEL Assay: An In Situ Method to Detect DNA Fragmentation in Apoptotic Cells

Published: April 30, 2023

Abstract

Source: Brannelly, L. A., et al. Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis. J. Vis. Exp. (2018).

In this video, the TUNEL assay is performed to detect the presence of apoptotic cells in a sample. The study involves using a special DNA polymerase – terminal deoxynucleotidyl transferase – that catalyzes the addition of labeled dUTPs to the 3' terminus of fragmented DNA, which can be observed and identified under a fluorescence microscope.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. TUNEL Assay Euthanize animals that are showing clinical signs of chytridiomycosis and an equal number of Bd-negative control animals. Dissect skin (dorsal, ventral, and thigh) samples from each animal. Fix the skin samples for 2 h in 4% v/v phosphate buffered formaldehyde. The short and consistent fixing time allows t…

Disclosures

The authors have nothing to disclose.

Materials

POLARstar Omega BMG Labtech Luminescent plate reader
384 well flat clear bottom plate Corning 3707
384 well low flange white flat bottom plate Corning 3570
Formal-Fixx 10% Neutral Buffered Formalin Fisher 6764254
ApopTag Red In Situ Apoptosis Detection Kit Merck Millipore S7165
PBS (Phosphate Buffered Saline),  pH 7.2 (1X) Thermo/Life 20-012-043
Parafilm Bemis PM996
Ethanol, 200 Proof, Molecular Grade Fisher BP2818500
ZEISS Axio Scan florescent miscroscope Carl Zeiss Florescent microscope
Cell culture petri plates Nunc 263991

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Cite This Article
Terminal Transferase-Mediated dUTP Nick End-Labeling—TUNEL Assay: An In Situ Method to Detect DNA Fragmentation in Apoptotic Cells. J. Vis. Exp. (Pending Publication), e21233, doi: (2023).

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