ProtocolI:TritonX-100LysisBuffer

In96flat-wellsplate,incubate4x106targetcells(40wellsof105perwell)withdesiredconcentrationofeffectors(105targetcellsperwell).Afterincubation,collectthecellsamplein1.5mlEppendorftube,spindown,resUSPendwith0.5mlPBSin1.5mleppendorftubes,andadd55uloflysisbufferfor20minonice(4oC).Centrifugetheeppendorftubesincoldat12,000gfor30minutes.Transferthesamplestonew1.5mleppendorftubesandthenextractthesupernatantwith1:1mixtureofphenol:chloroform(gentleagitationfor5minfollowedbycentrifugation)andprecipitateintwoequivalenceofcoldethanolandone-tenthequivalenceofsodiumacetate.Spindown,decant,andresuspendtheprecipitatesin30ulofdeionizedwater-RNasesolution(0.4mlwater+5ulofRNase)and5ulofloADIngbufferfor30minutesat37oC.Alsoinsert2ulofHindiIIIMarker(12ulofStockIV)ontheouterlanes.Runthe1.2%gelat5Vfor5minbeforeincreasingto100V.

ProtocolII:SDSLysisBufferAddSDSlysisbuffertotheincubatedcellsamples(preparedasinProtocolI).

StockI:TritonX-100LysisBuffer40mlof0.5MEDTA5mlof1MTrisClbufferpH8.05mlof100%TritonX-10050mlofH2O

StockII:SDSLysisBuffer

StockIII:1.2%AgaroseGel

Prepareastockof2literof1XTAE(i.e.,2liter+40mlof5XTAE).Add2.4gofagarosepower(1.2%agarose)to200mlof1XTAEsolutionandmicrowavefor4minathighpower.Thencoolthegelto50oCandadd25ulofethiumbromidebeforepouringitintothegelplate.Insertcombandletthegelpolymerized.

StockIV:HindiIIIMarker(50KblamdaDNA)4ulofHindiIIIMarker16ulofDeionizedWater4ulofLoadingBuffer

ProtocolII:DNAFragmentationAssayviaDipheylamine

In24-wellsplate,incubate5X106targetswithdesirednumberofeffectors.Afterincubation,transferthesamplesto15mltubes,centrifugefor30sat1500g,andresuspendin5mloflysisbuffer(StockIV)for15minonice.Centrifugethesamplesfor20minat27,000gtoseparatehigh-molecular-weightchromatinfromcleavageproducts.Resuspendthepelletin5mlofbuffer(stockV).Treatthesupernatantsandpelletswiththediphenylaminereagent(StockVI)andincubateat370Cfor16-24hrbeforecolorimetricassessment.

StockIV:LysisbufferatpH8.05mMTris-HCl20mMEDTA0.5%TritonX-100

StockV:BufferatpH8.010mMTris-HCl1mMEDTA

StockVI:Diphenylaminereagent(lightsensitive)1.5gofdiphenylamine(steam-distilled)100mlaceticacid(redistilled)1.5mlofconc.sulfuricacid

Onthedayofusage,add0.10mlofagacetaldehyde(16mg/ml)to20mlofthediphenylaminereagent.

ProtocolIII:DNAFragmentationvia3H-TdR

5X106targetcellswerelabeledwith50µlof3H-TdR(1mCi/ml)overnightin10mlofmedia.Thenextday,thecellswerewashed3Xwith10mlofPBSandincubatedin10mlofmediatochaseoutunincorporatedcytoplasmic3H-TdR.Afterincubatingfor2hrs,thecellswerewashed3XwithPBSandthenusedinlyticassayunderthesameconditionsasthe51Crreleaseassayin96v-wellplates.Attheendoftheassay,eachwellwastreatedwith20µlof1.0%Triton-Xonicefor5minutes,followedbycentrifugationat1500ginaBeckmanT-J6rotorfor15minutes.100µlofthesupernatantwereharvestedfromeachwellandcountedinascintillationcounter.Totalcountwasobtainedbyresuspendingthecellspriortoharvesting,andadding0.1%SDStosolublilizethecells.The%3Hreleasedwascalculatedwithanequationanalogoustothatfor%51Crreleased.