ASTM_E_2186_-_02a_2010.pdf

Designation:E218602a(Reapproved 2010)Standard Guide forDetermining DNA Single-Strand Damage in Eukaryotic CellsUsing the Comet Assay1This standard is issued under the fixed designation E2186;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This guide covers the recommended criteria for per-forming a single-cell gel electrophoresis assay(SCG)or Cometassay for the measurement of DNA single-strand breaks ineukaryotic cells.The Comet assay is a very sensitive methodfor detecting strand breaks in the DNA of individual cells.Themajority of studies utilizing the Comet assay have focused onmedical applications and have therefore examined DNA dam-age in mammalian cells in vitro and in vivo(1-4).2There isincreasing interest in applying this assay to DNA damage infreshwater and marine organisms to explore the environmentalimplications of DNA damage.1.1.1 The Comet assay has been used to screen the geno-toxicity 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).Usingthis method,significantly elevated levels of DNAdamage havebeen reported in cells collected from organisms at pollutedsites compared to reference sites(12-15).Studies have alsofound that increases in cellular DNA damage correspond withhigher order effects such as decreased growth,survival,anddevelopment,and correlate with significant increases in con-taminant body burdens(13,16).1.2 This guide presents protocols that facilitate the expres-sion of DNA alkaline labile single-strand breaks and thedetermination of their abundance relative to control or refer-ence cells.The guide is a general one meant to familiarize labpersonnel with the basic requirements and considerationsnecessary to perform the Comet assay.It does not containprocedures for available variants of this assay,which allow thedetermination of non-alkaline labile single-strand breaks ordouble-stranded DNA strand breaks(8),distinction betweendifferent cell types(13),identification of cells undergoingapoptosis(programmed cell death,(1,17),measurement ofcellular DNA repair rates(10),detection of the presence ofphotoactive DNA damaging compounds(14),or detection ofspecific DNA lesions(3,18).1.3 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory requirements prior to use.1.4 This guide is arranged as follows:SectionScope1Referenced Documents2Terminology3Summary of Guide4Significance and Use5Equipment and Reagents6Assay Procedures7Treatment of Data8Reporting Data9Keywords10AnnexA1References2.Referenced Documents2.1 ASTM Standards:3E1706 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater InvertebratesE1847 Practice for Statistical Analysis of Toxicity TestsConducted Under ASTM Guidelines3.Terminology3.1 The words“must,”“should,”“may,”“can,”and“might”have very specific meanings in this guide.“Must”is used toexpress the strongest possible recommendation,just short of anabsolute requirement.“Must”is only used in connection withfactors that relate directly to the acceptability of the test.“Should”is used to state that the specific condition is recom-mended and ought to be met if possible.Although violation ofon“should”is rarely a serious matter,the violation of severalwill often render the results questionable.Terms such as“is1This guide is under the jurisdiction ofASTM Committee E50 on EnvironmentalAssessment,Risk Management and CorrectiveActionand is the direct responsibilityof Subcommittee E50.47 on Biological Effects and Environmental Fate.Current edition approved March 1,2010.Published May 2010.Originallyapproved in 2002.Last previous edition approved 2002 as E218602A.DOI:10.1520/E2186-02AR10.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.For Annual Book of ASTMStandards volume information,refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 desirable,”“is often desirable,”and“might be desirable”areused in connection with less important factors.“May”is usedto mean“is(are)allowed to,”“can”is used to mean“is(are)able to,”and“might”is used to mean“could possibly.”Thusthe classic distinction between“may”and“can”is preservedand“might”is never used as a synonym for either“may”or“can.”3.2 Definitions:3.2.1 CCD camera,ncharge