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/**/

DESCRIPTIONofthemethod:TheDNAFragmentationAssayallowstodeterminetheamountofDNAthatisdegradedupontreatmentofcellswithcertainagents,e.g.withTNF-alphaoranti-Fasantibody(IPO-4).

Theprincipleofthisassayis:thecells"DNAisrADIoactivelylabeledbygrowingthecellsinpresenceof3H-Thymidinesothatradioactive3H-ThymidineisincorporatedintotheDNA.ThenthelabeledcellsareincubatedwithorwithouttheDNAfragmentationinducingagent.Duringthisincubationtheaddedagent(e.g.TNF-alphainducescellstodiebyapoptosisandconsequentlythefragmentationofDNAwhiletheDNAofuntreatedcellsremainsintact.Afterincubation(e.g.48h)thecellsareharvested:duringharvesting,thecellsarewashedoutofthewellsofthe96wellplatewithbidestwater:thecellsandorganellesburstandthecell"sDNAissetfree.ThecellfragmentsandDNAarepassedthroughafiltermembrane(glassfiber).Onlyparticlesofsmallerthan1,5祄canpassthefilter.So,intactDNA(withafragmentlengthintherangeofmilimetersorevencentimeters)willnotbeabletopassthefilterbutbecollectedonthefiltermembrane.DNAthatwascleaved/degradedintofragmentsofabout5000bporlesswillbesmallenoughtopasstheporesofthefilterandwon"tbecollectedonthefilter.Thefiltermembraneisdryedandtheamountofradioactivity(whatcorrespondstotheamountofintactDNA)countedinascintillationcounter.TocalculatethepercentageofDNAfragmentationyoucomparethecountedradioactivity(countsperminute=cpm)ofcellsthatwerenottreatedwiththecpmincellsthatweretreatedwithagent:

Fragmentation[%]=[cpm(untreated)-cpm(treated)]/cpm(untreated).

PROCEDURE

Ina25cm2flask:incubatecells(about200000cells)for16h-24hinthepresenceof2礐i/ml3H-Thymidinein5mlRPMIcompletemedium(i.e.10祃of1礐i/祃stocksolution);
Harvestcells(washtwotimeswithRPMI),countcells
Seed3500-5000cellsperwellsin96wellplate(inavolumeof100祃);
addagent(e.g.TNF-alphainavolumeof100祃RPMIorjust100祃RPMI
Incubateplateforacertaintime(e.g.48h)at37癈;
Harvestcells:pipetsupernatantintoanotherplate,pipet50祃ofTrypsine/EDTAtothecells,incubate37癈untilallcellsaredetached,pipetthesupernatantsbacktothecorrespondingwellscontainingthedetachedcells,harvestwithharvester;
Addscintillationfluidtothedriedfilterdiscsandcounttheradioacivitywiththebeta-counter;
CalculateDNAFragmentation.

CAUTION:Disposeradioactivematerialsintoradioactivewastecontainers!!!

【求助】蛋白加上样缓冲液煮后有沉淀生成 蛋白质和糖学技术讨论...

【求助】蛋白加上样缓冲液煮后有沉淀生成蛋白质和糖学技术讨论...