0065-1281/02/104/04-367$15.00/0ComparisonoftheTUNEL,laminBandannexinVmethodsforthedetectionofapoptosisbyflowcytometryDanaKylarová1*,JirˇinaProcházková1,JanaMad’arová2,JanBartosˇ3,andVáclavLichnovsk’y11DepartmentofHistologyandEmbryologyand2DepartmentofPathology,FacultyofMedicine,PalackyUniversity,and3InstituteofExperimentalBotany,AcademyofSciencesoftheCzechRepublic,Olomouc,CzechRepublicReceived19February2002;accepted29April2002SummaryThepresentstudywasperformedinordertocomparethesensitivityof3apoptosis-detectionmethodsusingamodelcellcultureafterinductionofapoptosis.Evaluationwasperformedbymeansofflowcytometryandlightmicroscopy.ItappearedthatbothTUNELandannexinVmethodsaresensitiveandspecificandproducedsimilardatainallmeasurements.TheimmunocytochemicaldetectionoflaminBwaslessreliablethantheothermethods.Keywords:apoptosis–flowcytometry–TUNEL–annexinV–laminBIntroductionApoptosisisthemostintensivelystudiedformofpro-grammedcelldeathandischaracterizedbyspecificbio-chemicalandmorphologicalchanges,whichcanbedetectedbymanymethods.Morphologicalchangestakingplaceduringthepro-cessofapoptosishavebeendescribed,firstofall,byKerretal.(1972).NuclearchangestakingplaceduringapoptosisarebasedonregularDNAcleavageintospe-cificfragmentsthataredetectablebyTdT-mediateddUTPnickendlabelling(TUNEL).ThemethodisbasedonthetransferoflabelleddNTPto3´-OHendsofDNAfragmentsbyterminaldeoxynucleotidyltrans-ferase(Gavrielietal.,1992).Thelossofmanystruc-turalproteinssuchaslaminB(Weaveretal.,1996),causedbycaspaseactivityduringapoptosis,isanothermarkerinanumberofapoptosis-detectingmethods.Alterationsincellmembranessuchasadecreaseinthemembranepotentialofapoptoticcellswhilethemem-branesarekeptintactareoftenusedinflowcytometrytodetectapoptosis(Darzynckiewiczetal.,1998).Anotheralterationinmembranesofapoptoticcellsistheexpositionofphosphatidylserineatthemembranesurface,whichisasignalforphagocytosisofapoptoticcells.ThismoleculeselectivelybindsannexinVashasbeendescribedbyKoopmanetal.(1994).Noneofthe...
