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TUNEL assay as a measure of chemotherapy-induced apoptosis
1University of Medicine & Dentistry of New Jersey, Division Medical Oncology/Hematology, Newark, USA.
Abstract:
Chemotherapy induces injury to tumor cells, which subsequently die by a number of processes. One of those processes is apoptosis, and its measurement can be a useful tool to understanding the mechanisms of action of chemotherapy agents, drug resistance, and tumor biology. Cells undergoing apoptosis eventually cleave their DNA with nucleases in a characteristic pattern that leaves free 3'OH ends. This permits the identification of cells undergoing apoptosis by transfer of fluorescence-labeled dUTP to the free 3'OH ends using the enzyme terminal deoxynucleotidyl transferase (TdT) in a process termed terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL). This chapter outlines the concepts governing the measurement of apoptosis following chemotherapy by TUNEL in cells in culture and in tissue biopsied from patients or laboratory animals that received chemotherapy. It lists the reagents, procedures for cell and tissue handling, labeling techniques, caveats, and appropriate controls for labeling 3'OH termini with TdT and quantifying the results.
Insights
Chemotherapy triggers apoptosis, a programmed cell death pathway. The TUNEL assay detects this process by labeling DNA strand breaks, aiding chemotherapy research and tumor biology studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Chemotherapy induces tumor cell death through various mechanisms.
- Apoptosis is a key process in chemotherapy's efficacy.
- Measuring apoptosis provides insights into chemotherapy mechanisms, drug resistance, and tumor biology.
Purpose of the Study:
- To outline the concepts and methods for measuring chemotherapy-induced apoptosis.
- To detail the application of the TUNEL assay for apoptosis detection.
- To provide guidance on TUNEL assay implementation in cell culture and tissue samples.
Main Methods:
- Utilizing the terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL) assay.
- Labeling free 3'OH DNA ends generated during apoptosis with fluorescently labeled dUTP.
- Employing the enzyme terminal deoxynucleotidyl transferase (TdT) for labeling.
Main Results:
- The TUNEL assay enables the identification of cells undergoing apoptosis.
- This method allows for the quantification of apoptosis in response to chemotherapy.
- The chapter provides a comprehensive guide to TUNEL assay procedures and controls.
Conclusions:
- Apoptosis measurement via TUNEL is a valuable tool in chemotherapy research.
- Understanding apoptosis mechanisms is crucial for developing effective cancer treatments.
- The TUNEL assay facilitates the analysis of chemotherapy effects in both in vitro and in vivo models.
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