ASTM E2186-2002a(2010) 用彗星分析法测定真核细胞中DNA单线损伤的标准指南
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【英文标准名称】:StandardGuideforDeterminingDNASingle-StrandDamageinEukaryoticCellsUsingtheCometAssay
【原文标准名称】:用彗星分析法测定真核细胞中DNA单线损伤的标准指南
【标准号】:ASTME2186-2002a(2010)
【标准状态】:现行
【国别】:美国
【发布日期】:2002
【实施或试行日期】:
【发布单位】:美国材料与试验协会(US-ASTM)
【起草单位】:E47.02
【标准类型】:(Guide)
【标准水平】:()
【中文主题词】:
【英文主题词】:biomarker;cellular;Cometassay;DNAdamage;DNAstrandbreaks;stresseffects;toxic;Eukaryoticcells;Single-cellgel(SCG)electrophoresisassay;Single-strandDNAdamage;Stress--environmentaleffects;Biomarker;Cometassay;DNA(deoxyribonuc
【摘要】:AcommonresultofcellularstressisanincreaseinDNAdamage.DNAdamagemaybemanifestintheformofbasealterations,adductformation,strandbreaks,andcrosslinkages(19).Strandbreaksmaybeintroducedinmanyways,directlybygenotoxiccompounds,throughtheinductionofapoptosisornecrosis,secondarilythroughtheinteractionwithoxygenradicalsorotherreactiveintermediates,orasaconsequenceofexcisionrepairenzymes(20-22).Inadditiontoalinkagewithcancer,studieshavedemonstratedthatincreasesincellularDNAdamageprecedeorcorrespondwithreducedgrowth,abnormaldevelopment,andreducedsurvivalofadults,embryos,andlarvae(16,23,24).TheCometassaycanbeeasilyutilizedforcollectingdataonDNAstrandbreakage(9,25,26).Itisasimple,rapid,andsensitivemethodthatallowsthecomparisonofDNAstranddamageindifferentcellpopulations.Aspresentedinthisguide,theassayfacilitatesthedetectionofDNAsinglestrandbreaksandalkalinelabilesitesinindividualcells,andcandeterminetheirabundancerelativetocontrolorreferencecells(9,16,26).Theassayoffersanumberofadvantages;damagetotheDNAinindividualcellsismeasured,onlyextremelysmallnumbersofcellsneedtobesampledtoperformtheassay(x003C;10000),theassaycanbeperformedonpracticallyanyeukaryoticcelltype,andithasbeenshownincomparativestudiestobeaverysensitivemethodfordetectingDNAdamage(2,27).Thesearegeneralguidelines.Therearenumerousproceduralvariantsofthisassay.Thevariationusedisdependentuponthetypeofcellsbeingexamined,thetypesofDNAdamageofinterest,andtheimagingandanalysiscapabilitiesofthelabconductingtheassay.TovisualizetheDNA,itisstainedwithafluorescentdye,orforlightmicroscopeanalysistheDNAcanbesilverstained(28).Onlyfluorescentstainingmethodswillbedescribedinthisguide.ThemicroscopicdeterminationofDNAmigrationcanbemadeeitherbyeyeusinganocularmicrometerorwiththeuseofimageanalysissoftware.Scoringbyeyecanbeperformedusingacalibratedocularmicrometerorbycategorizingcellsintofourtofiveclassesbasedontheextentofmigration(29,30).ImageanalysissystemsarecomprisedofaCCDcameraattachedtoafluorescentmicroscopeandsoftwareandhardwaredesignedspecificallytocaptureandanalyzeimagesoffluorescentlystainednuclei.Usingsuchasystem,itispossibletomeasurethefluorescenceintensityanddistributionofDNAinandawayfromthenucleus(8).Usingdifferentproceduralvariants,theassaycanbeutilizedtomeasurespecifictypesofDNAalterationsandDNArepairactivity(1,3,8,10,13,14,17,18).Alkalinelysisandelectrophoresisconditionsareusedforthedetectionofsingle-strandedDNAdamage,whereasneutralpHconditionsfacilitatethedetectionofdouble-strandbreaks(31).VarioussampletreatmentscanbeusedtoexpressspecifictypesofDNAdamage,orasinonemethod,topreservestranddamageatsitesofDNArepair(10).Nucleasedigestionstepscanbeusedtointroducestrandbreaksatspecificlesionsites.Usingthisapproach,oxidativebasedamagecanbedetectedbytheuseofendonucleaseIII(18),aswellasDNAmodificationsresultingfromexposuretoultravioletlight(UV)throughtheuseofT4endonucleaseV(3).Modificationsofthistypevastlyexpandtheutilityofthisassayandaregoodexamplesofitsversatility.Asufficientknowledgeofthebiologyofcel.......
【中国标准分类号】:C05
【国际标准分类号】:07_100_01
【页数】:10P.;A4
【正文语种】:英语
【原文标准名称】:用彗星分析法测定真核细胞中DNA单线损伤的标准指南
【标准号】:ASTME2186-2002a(2010)
【标准状态】:现行
【国别】:美国
【发布日期】:2002
【实施或试行日期】:
【发布单位】:美国材料与试验协会(US-ASTM)
【起草单位】:E47.02
【标准类型】:(Guide)
【标准水平】:()
【中文主题词】:
【英文主题词】:biomarker;cellular;Cometassay;DNAdamage;DNAstrandbreaks;stresseffects;toxic;Eukaryoticcells;Single-cellgel(SCG)electrophoresisassay;Single-strandDNAdamage;Stress--environmentaleffects;Biomarker;Cometassay;DNA(deoxyribonuc
【摘要】:AcommonresultofcellularstressisanincreaseinDNAdamage.DNAdamagemaybemanifestintheformofbasealterations,adductformation,strandbreaks,andcrosslinkages(19).Strandbreaksmaybeintroducedinmanyways,directlybygenotoxiccompounds,throughtheinductionofapoptosisornecrosis,secondarilythroughtheinteractionwithoxygenradicalsorotherreactiveintermediates,orasaconsequenceofexcisionrepairenzymes(20-22).Inadditiontoalinkagewithcancer,studieshavedemonstratedthatincreasesincellularDNAdamageprecedeorcorrespondwithreducedgrowth,abnormaldevelopment,andreducedsurvivalofadults,embryos,andlarvae(16,23,24).TheCometassaycanbeeasilyutilizedforcollectingdataonDNAstrandbreakage(9,25,26).Itisasimple,rapid,andsensitivemethodthatallowsthecomparisonofDNAstranddamageindifferentcellpopulations.Aspresentedinthisguide,theassayfacilitatesthedetectionofDNAsinglestrandbreaksandalkalinelabilesitesinindividualcells,andcandeterminetheirabundancerelativetocontrolorreferencecells(9,16,26).Theassayoffersanumberofadvantages;damagetotheDNAinindividualcellsismeasured,onlyextremelysmallnumbersofcellsneedtobesampledtoperformtheassay(x003C;10000),theassaycanbeperformedonpracticallyanyeukaryoticcelltype,andithasbeenshownincomparativestudiestobeaverysensitivemethodfordetectingDNAdamage(2,27).Thesearegeneralguidelines.Therearenumerousproceduralvariantsofthisassay.Thevariationusedisdependentuponthetypeofcellsbeingexamined,thetypesofDNAdamageofinterest,andtheimagingandanalysiscapabilitiesofthelabconductingtheassay.TovisualizetheDNA,itisstainedwithafluorescentdye,orforlightmicroscopeanalysistheDNAcanbesilverstained(28).Onlyfluorescentstainingmethodswillbedescribedinthisguide.ThemicroscopicdeterminationofDNAmigrationcanbemadeeitherbyeyeusinganocularmicrometerorwiththeuseofimageanalysissoftware.Scoringbyeyecanbeperformedusingacalibratedocularmicrometerorbycategorizingcellsintofourtofiveclassesbasedontheextentofmigration(29,30).ImageanalysissystemsarecomprisedofaCCDcameraattachedtoafluorescentmicroscopeandsoftwareandhardwaredesignedspecificallytocaptureandanalyzeimagesoffluorescentlystainednuclei.Usingsuchasystem,itispossibletomeasurethefluorescenceintensityanddistributionofDNAinandawayfromthenucleus(8).Usingdifferentproceduralvariants,theassaycanbeutilizedtomeasurespecifictypesofDNAalterationsandDNArepairactivity(1,3,8,10,13,14,17,18).Alkalinelysisandelectrophoresisconditionsareusedforthedetectionofsingle-strandedDNAdamage,whereasneutralpHconditionsfacilitatethedetectionofdouble-strandbreaks(31).VarioussampletreatmentscanbeusedtoexpressspecifictypesofDNAdamage,orasinonemethod,topreservestranddamageatsitesofDNArepair(10).Nucleasedigestionstepscanbeusedtointroducestrandbreaksatspecificlesionsites.Usingthisapproach,oxidativebasedamagecanbedetectedbytheuseofendonucleaseIII(18),aswellasDNAmodificationsresultingfromexposuretoultravioletlight(UV)throughtheuseofT4endonucleaseV(3).Modificationsofthistypevastlyexpandtheutilityofthisassayandaregoodexamplesofitsversatility.Asufficientknowledgeofthebiologyofcel.......
【中国标准分类号】:C05
【国际标准分类号】:07_100_01
【页数】:10P.;A4
【正文语种】:英语
下载地址: 点击此处下载