实验技术相关名词

1.Fluorescence-activatedcellsorting(FACS)Fluorescence-activatedcellsorting(FACS)isaspecializedtypeofflowcytometry.Itprovidesamethodforsortingaheterogeneousmixtureofbiologicalcellsintotwoormorecontainers,onecellatatime,baseduponthespecificlightscatteringandfluorescentcharacteristicsofeachcell.Itisausefulscientificinstrumentasitprovidesfast,objectiveandquantitativerecordingoffluorescentsignalsfromindividualcellsaswellasphysicalseparationofcellsofparticularinterest.ThetechniquewasexpandedbyLenHerzenberg,whowasresponsibleforcoiningthetermFACS.[22]HerzenbergwontheKyotoPrizein2006forhisseminalworkinflowcytometry.Thecellsuspensionisentrainedinthecenterofanarrow,rapidlyflowingstreamofliquid.Theflowisarrangedsothatthereisalargeseparationbetweencellsrelativetotheirdiameter.Avibratingmechanismcausesthestreamofcellstobreakintoindividualdroplets.Thesystemisadjustedsothatthereisalowprobabilityofmorethanonecellperdroplet.Justbeforethestreambreaksintodroplets,theflowpassesthroughafluorescencemeasuringstationwherethefluorescentcharacterofinterestofeachcellismeasured.Anelectricalchargingringisplacedjustatthepointwherethestreambreaksintodroplets.Achargeisplacedontheringbasedontheimmediatelypriorfluorescenceintensitymeasurement,andtheoppositechargeistrappedonthedropletasitbreaksfromthestream.Thechargeddropletsthenfallthroughanelectrostaticdeflectionsystemthatdivertsdropletsintocontainersbasedupontheircharge.Insomesystems,thechargeisapplieddirectlytothestream,andthedropletbreakingoffretainschargeofthesamesignasthestream.Thestreamisthenreturnedtoneutralafterthedropletbreaksoff.TheacronymFACSistrademarkedandownedbyBecton,DickinsonandCompany.[23]Amongthelargemajorityofresearcherswhousethistechnologyforsortingoranalysis,thistermhasbecomegenericized,muchlikeXeroxforphotocopyorKleenexforfacialtissue.2.TUNELassayTerminaldeoxynucleotidyltransferasedUTPnickendlabeling(TUNEL)isamethodfordetectingDNAfragmentationbylabelingtheterminalendofnucleicacidsMethodTUNELisacommonmethodfordetectingDNAfragmentationthatresultsfromapoptoticsignalingcascades.[2]TheassayreliesonthepresenceofnicksintheDNAwhichcanbeidentifiedbyterminaldeoxynucleotidyltransferaseorTdT,anenzymethatwillcatalyzetheadditionofdUTPsthataresecondarilylabeledwithamarker.ItmayalsolabelcellsthathavesufferedsevereDNAdamage.HistoryOriginallydescribedinthepaperbyGavrieli,Sherman,andBen-Sassonin1992,[3]TUNELhasbecomeoneofthemainmethodsfordetectingapoptoticprogrammedcelldeath.However,foryearstherehasbeenadebateaboutitsaccuracy,duetoproblemsintheoriginalassaywhichcausednecroticcellstobeinappropriatelylabeledasapoptotic.[4]Themethodhassubsequentlybeenimproveddramaticallyandifperformedcorrectlyshouldonlyidentifycellsinthelastphaseofapoptosis.[5][6]NewmethodsincorporatethedUTPsmodifiedbyfluorophoresorhaptens,includingbiotinorbromine,whichcanbedetecteddirectlyinthecaseofafluorescently-modifiednucleotide(i.e.,fluorescein-dUTP),orindirectlywithstreptavidinorantibodies,ifbiotin-dUTPorBrdUTPareused,respectively.Oftenatlatestagesofapoptosis,adherentcellsareknowntodetachor“pop”off.ForareliableandreproducibleTUNELimagingassay,themodifiednucleotidemustnotonlybeanacceptablesubstrateforTdT,butthedetectionmethodmustalsobesensitivewithoutbringingaboutanyadditionallossofcellsfromthesample.