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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
Published on: April 24, 2011
An unusual artefact in the terminal deoxynucleotidyl transferase assay for apoptotic cells
S Detre1, M G Ormerod, J C Titley
1Academic Department of Biochemistry, Royal Marsden NHS Trust, London, United Kingdom.
Abstract:
A staining artefact associated with the terminal deoxynucleotidyl assay for apoptotic cells is described. the Artefact is caused by particulate matter in the reconstituted dried milk used in the washing buffer. We recommend filtering the washing buffer before use.
Insights
A staining artifact in the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptotic cells is caused by milk particulates. Filtering the washing buffer prevents this artifact, ensuring accurate apoptosis detection.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay is a common method for detecting apoptotic cells.
- Accurate detection of apoptosis is crucial in various research fields, including cancer biology and drug development.
Purpose of the Study:
- To identify the cause of a specific staining artifact observed during the TUNEL assay.
- To propose a simple method to prevent this artifact and improve assay reliability.
Main Methods:
- The study involved performing the TUNEL assay under standard conditions.
- Investigated the composition of reagents used in the washing buffer, specifically reconstituted dried milk.
- Microscopic examination of stained cells to identify the nature of the artifact.
Main Results:
- A consistent staining artifact was observed in cells stained using the TUNEL assay.
- The artifact was traced to the presence of particulate matter originating from reconstituted dried milk in the washing buffer.
- Filtering the washing buffer effectively removed the particulate matter and eliminated the staining artifact.
Conclusions:
- Particulate matter in reconstituted dried milk is a source of staining artifacts in the TUNEL assay.
- Filtering the washing buffer is a simple and effective method to prevent this artifact.
- Implementing this filtration step can enhance the accuracy and reliability of apoptosis detection using the TUNEL assay.
