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 |
Tags
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).