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ASTM E 2186 : 2002 : REV A : R2016

Superseded

Superseded

A superseded Standard is one, which is fully replaced by another Standard, which is a new edition of the same Standard.

View Superseded by

Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay

Available format(s)

Hardcopy , PDF

Superseded date

29-12-2023

Language(s)

English

Published date

27-05-2016

$122.77
Including GST where applicable

CONTAINED IN VOL. 11.06, 2016 Defines the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells.

Committee
E 50
DocumentType
Guide
Pages
10
ProductNote
Reconfirmed 2016
PublisherName
American Society for Testing and Materials
Status
Superseded
SupersededBy
Supersedes

1.1This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The Comet assay is a very sensitive method for detecting strand breaks in the DNA of individual cells. The majority of studies utilizing the Comet assay have focused on medical applications and have therefore examined DNA damage in mammalian cells in vitro and in vivo (1-4).2 There is increasing interest in applying this assay to DNA damage in freshwater and marine organisms to explore the environmental implications of DNA damage.

1.1.1The Comet assay has been used to screen the genotoxicity of a variety of compounds on cells in vitro and in vivo (5-7), as well as to evaluate the dose-dependent anti-oxidant (protective) properties of various compounds (3, 8-11). Using this method, significantly elevated levels of DNA damage have been reported in cells collected from organisms at polluted sites compared to reference sites (12-15). Studies have also found that increases in cellular DNA damage correspond with higher order effects such as decreased growth, survival, and development, and correlate with significant increases in contaminant body burdens (13, 16).

1.2This guide presents protocols that facilitate the expression of DNA alkaline labile single-strand breaks and the determination of their abundance relative to control or reference cells. The guide is a general one meant to familiarize lab personnel with the basic requirements and considerations necessary to perform the Comet assay. It does not contain procedures for available variants of this assay, which allow the determination of non-alkaline labile single-strand breaks or double-stranded DNA strand breaks (8), distinction between different cell types (13), identification of cells undergoing apoptosis (programmed cell death, (1, 17)), measurement of cellular DNA repair rates (10), detection of the presence of photoactive DNA damaging compounds (14), or detection of specific DNA lesions (3, 18) .

1.3This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.

1.4This guide is arranged as follows:

Section

Scope

1

Referenced Documents

2

Terminology

3

Summary of Guide

4

Significance and Use

5

Equipment and Reagents

6

Assay Procedures

7

Treatment of Data

8

Reporting Data

9

Keywords

10

Annex

Annex A1

References

ASTM F 3294 : 2018 Standard Guide for Performing Quantitative Fluorescence Intensity Measurements in Cell-based Assays with Widefield Epifluorescence Microscopy
ASTM F 1439 : 2003 : R2018 Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials

ASTM E 1706 : 2005 Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates
ASTM E 1847 : 1996 : R2008 Standard Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines
ASTM E 1706 : 2000 : EDT 1 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Fresh Water Invertebrates
ASTM E 1706 : 2020 Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates
ASTM E 1706 : 2000 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Fresh Water Invertebrates
ASTM E 1706 : 2005 : EDT 1 Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates
ASTM E 1847 : 1996 : R2003 Standard Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines
ASTM E 1847 : 1996 Standard Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines
ASTM E 1706 : 2005 : R2010 Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates (Withdrawn 2019)
ASTM E 1706 : 2000 : EDT 2 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates
ASTM E 1706 : 2004 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates
ASTM E 1847 : 1996 : R2013 Standard Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines (Withdrawn 2022)
ASTM E 1706 : 2019 Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates

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