Detection of BrdU Incorporation in DNA Synthesizing Cells

NOTE:Bromodeoxyuridineisaknowncarcinogen.Propidiumiodine(PI)isknowntobetoxicandcarcinogenic. 1. Pulseactivelygrowingcellsinatissuecultureflaskforonehourwith10礛BrdU(Sigma,Cat.No.B5002). 2. Pourcontentsoftissuecultureflaskintoacentrifugetube.Centrifuge10minutesat400xg(allcentrifugationsteps)areperformedat400xg,atRT).Aspiratesupernatant.Loosenpelletbytappingtube. 3. Whilevortexing,addicecold70%ethanoltocells,dropwise,toafinalconcentrationof1x106cells/100祃.Incubate20minutesatRT. 4. Aliquot100祃intoeachtesttube(12mmx75mm).Washwith1mlwashbuffer.Centrifuge5minutes.Aspiratesupernatant.Loosenpellet. 5. ResUSPendpelletindenaturingsolution.Mixwell.Incubate20minutesatRT.NOTE:Denaturingsolutionmustbemadefresh. 6. Add1mlwashbuffer.Mixwell.Centrifuge5minutes.Aspiratesupernatant. 7. Resuspendpelletin0.5ml0.1Msodiumborate(Na2B4O7),pH8.5,toneutralizeanyresidualacid.Incubate2minutesatRT. 8. Add1mlwashbuffer.Mixwell.Centrifuge5minutes.Aspiratesupernatant. 9. Addprimaryantibody:diluteanti-BrdUmonoclonalanitbody(PharmingenCat.No.33281A)indilutionbuffer,suchthat50祃containstheoptimalconcentration.Resuspendcellpelletin50祃ofthedilutedantibody.Incubate20minutesatRT. 10. Add1mlwashbuffer.Mixwell.Centrifuge5minutes.Aspiratesupernatant. 11. Addsecondaryantibody:diluteFITC-conjugatedgoatanti-mouseIg(PharMingenCat.No.12064D)indilutionbuffer,suchthat50祃containstheoptimalconcentration.Resuspendcellpelletin50祃ofthedilutedantibody.Incubate20minutesatRT.NOTE:EliminatethisstepwhenusingFITC-conjugatedanti-BrdU(Cat.No.33284X) 12. Add1mlwashbuffer.Mixwell.Centrifuge5minutes.Aspiratesupernatant. 13. Resuspendpelletin0.5mlpropidiumiodide(10礸/mlinPBS).Incubate30minutesatRT,protectedfromlight. 14. Analyzethecellsbyflowcytometry,excitingat488nmandmeasuringtheBrdU-linkedgreenfluorescence(FITC)througha514nmbandpassfilterandtheDNSlinkedredfluorescence(PI)througha600nmwave-lengthfilter. 15. Followinganalysis,flushflowcytometerfor10minuteswith10%bleachand5minuteswithdH2O. SOLUTIONS:WashingSolution:PBScontaining0.5%BSADenaturingSolution:2MHClDilutionBuffer:PBScontaining0.5%Tween?20,0.5%BSA