线粒体荧光探针信息大全.docx

线粒体荧光探针信息大全.docx

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线粒体荧光探针信息大全

线粒体荧光探针信息大全（ProbesforMitochondria）包括各种常用探针，如JC-1，JC-9，TMRM，TMRE等

Mitochondriaarefoundineukaryoticcells,wheretheymakeupasmuchas10%ofthecellvolume.Theyarepleomorphicorganelleswithstructuralvariationsdependingoncelltype,cell-cyclestageandintracellularmetabolicstate.Thekeyfunctionofmitochondriaisenergyproductionthroughoxidativephosphorylation（OxPhos）andlipidoxidation.1,2Severalothermetabolicfunctionsareperformedbymitochondria,includingureaproductionandheme,non-hemeironandsteroidbiogenesis,aswellasintracellularCa2+homeostasis.Mitochondriaalsoplayapivotalroleinapoptosis—aprocessbywhichunneededcellsareremovedduringdevelopment,anddefectivecellsareselectivelydestroyedwithoutsurroundingorganelledamageinsomatictissues3–5（Section15.5）.Formanyofthesemitochondrialfunctions,thereisonlyapartialunderstandingofthecomponentsinvolved,withevenlessinformationonmechanismandregulation.

VisualizingMitochondriainCellsandTissues

Themorphologyofmitochondriaishighlyvariable.Individingcells,theorganellecanswitchbetweenafragmentedmorphologywithmanyovoid-shapedmitochondria,asoftenshownintextbooks,andareticuluminwhichtheorganelleisasingle,many-branchedstructure.Thecellcycle–andmetabolicstate–dependentchangesinmitochondrialmorphologyarecontrolledbyasetofproteinsthatcausefissionandfusionoftheorganellemass.Mutationsintheseproteinsarethecauseofseveralhumandiseases,indicatingtheimportanceofoverallmorphologyforcellfunctioning（seeNote12.2"TechnicalFocus:

MitochondriainDiseases"）.Organellemorphologyisalsocontrolledbycytoskeletalelements,includingactinfilamentsandmicrotubules.Innondividingtissue,overallmitochondrialmorphologyisverycelldependent,withmitochondriaspiralingaroundtheaxonemeinspermatozoa,andovoidbandsofmitochondriaintercalatingbetweenactomyosinfilaments.Thereisemergingevidenceoffunctionallysignificantheterogeneityofmitochondrialformswithinindividualcells.

Theabundanceofmitochondriavarieswithcellularenergylevelandisafunctionofcelltype,cell-cyclestageandproliferativestate.Forexample,brownadiposetissuecells,6hepatocytes7andcertainrenalepithelialcells8tendtoberichinactivemitochondria,whereasquiescentimmune-systemprogenitororprecursorcellsshowlittlestainingwithmitochondrion-selectivedyes.9ThenumberofmitochondriaisreducedinAlzheimer'sdiseaseandtheirproteinandnucleicacidsareaffectedbyreactiveoxygenspecies,includingnitricoxide10（Chapter18）.

MolecularProbeshasarangeofmitochondrion-selectivedyeswithwhichtomonitormitochondrialmorphologyandorganellefunctioning.Theuptakeofmostmitochondrion-selectivedyesisdependentonthemitochondrialmembranepotential;nonylacridineorangeandpossiblyourMitoTrackerGreenFM,MitoFluorGreenandMitoFluorRed589probesarenotableexceptions,althoughtheirmembranepotential–independentuptakeandfluorescencehasbeenquestionedinsomecelltypes.11,12Mitochondrion-selectivereagentsenableresearcherstoprobemitochondrialactivity,localizationandabundance,13,14aswellastomonitortheeffectsofsomepharmacologicalagents,suchasanestheticsthataltermitochondrialfunction.15MolecularProbesoffersavarietyofcell-permeantstainsformitochondria,aswellassubunit-specificmonoclonalantibodiesdirectedagainstproteinsintheoxidativephosphorylation（OxPhos）system,allofwhicharediscussedbelow.

MitoTrackerProbes:

FixableMitochondrion-SelectiveProbes

Althoughconventionalfluorescentstainsformitochondria,suchasrhodamine123andtetramethylrosamine,arereadilysequesteredbyfunctioningmitochondria,theyaresubsequentlywashedoutofthecellsoncethemitochondrion'smembranepotentialislost.Thischaracteristiclimitstheiruseinexperimentsinwhichcellsmustbetreatedwithaldehyde-basedfixativesorotheragentsthataffecttheenergeticstateofthemitochondria.Toovercomethislimitation,MolecularProbeshasdevelopedMitoTrackerprobes—aseriesofpatentedmitochondrion-selectivestainsthatareconcentratedbyactivemitochondriaandwellretainedduringcellfixation.16BecausetheMitoTrackerOrange,MitoTrackerRedandMitoTrackerDeepRedprobesarealsoretainedfollowingpermeabilization,thesampleretainsthefluorescentstainingpatterncharacteristicoflivecellsduringsubsequentprocessingstepsforimmunocytochemistry,insituhybridizationorelectronmicroscopy.Inaddition,MitoTrackerreagentseliminatesomeofthedifficultiesofworkingwithpathogeniccellsbecause,oncethemitochondriaarestained,thecellscanbetreatedwithfixativesbeforethesampleisanalyzed.

PropertiesofMitoTrackerProbes

MitoTrackerprobesarecell-permeantmitochondrion-selectivedyesthatcontainamildlythiol-reactivechloromethylmoiety.Thechloromethylgroupappearstoberesponsibleforkeepingthedyeassociatedwiththemitochondriaafterfixation.Tolabelmitochondria,cellsaresimplyincubatedinsubmicromolarconcentrationsoftheMitoTrackerprobe,whichpassivelydiffusesacrosstheplasmamembraneandaccumulatesinactivemitochondria.Oncetheirmitochondriaarelabeled,thecellscanbetreatedwithaldehyde-basedfixativestoallowfurtherprocessingofthesample;withtheexceptionofMitoTrackerGreenFM,subsequentpermeabilizationwithcoldacetonedoesnotappeartodisturbthestainingpatternoftheMitoTrackerdyes.

MolecularProbesofferssevenMitoTrackerreagentsthatdifferinspectralcharacteristics,oxidationstateandfixability（Table12.2）.MitoTrackerprobesareprovidedinspeciallypackagedsetsof20vials,eachcontaining50µgforreconstitutionasrequired.

Orange-,Red-andInfrared-FluorescentMitoTrackerDyes

WeofferMitoTrackerderivativesoftheorange-fluorescenttetramethylrosamine（MitoTrackerOrangeCMTMRos,M7510;Figure12.3）andthered-fluorescentX-rosamine（MitoTrackerRedCMXRos,M7512;Figure12.4）,aswellasournewestderivatives,theMitoTrackerRed580andMitoTrackerDeepRed633probes（M22425,M22426;Figure12.5,Figure12.6）.BecausetheMitoTrackerRedCMXRos,MitoTrackerRed580andMitoTrackerDeepRed633probesproducelonger-wavelengthfluorescencethatiswellresolvedfromthefluorescenceofgreen-fluorescentdyes,theyaresuitableformulticolorlabelingexperiments（Figure1.45,Figure8.7,Figure12.7,Figure12.8,Figure12.9）,includingthosethatemployimagedeconvolutiontechniques（seeNote12.3"TechnicalFocus:

Wide-FieldDeconvolutionMicroscopy"）.Alsoavailablearechemicallyreducedformsofthetetramethylrosamine（MitoTrackerOrangeCM-H2TMRos,M7511;Figure12.10）andX-rosamine（MitoTrackerRedCM-H2XRos,M7513;Figure12.11）MitoTrackerprobes.UnlikeMitoTrackerOrangeCMTMRosandMitoTrackerRedCMXRos,thereducedversionsoftheseprobesdonotfluoresceuntiltheyenteranactivelyrespiringcell,wheretheyareoxidizedtothefluorescentmitochondrion-selectiveprobeandthensequesteredinthemitochondria（Figure12.12,Figure12.50,Figure15.13）.TheMitoTrackerprobeshaveprovenusefulfor:

∙Assayingtheroleofakinesin-likeproteinongermplasmaggregationinXenopusoocytes17

∙Detectingearlyapoptosis（Section15.5）,whichismarkedbyadisruptionofmitochondrialtransmembranepotentialinallcelltypesstudied18–20

∙Determiningthemechanismbywhichmitochondrialshapeisestablishedandmaintainedinyeast21

∙Examiningthetimecourseofcellswellinginahumancollecting-ductcelllineusingtotalinternalreflection（TIR）microfluorimetry22

∙Localizinganovelkinesinmotorproteininvolvedintransportofmitochondriaalongmicrotubules23

∙Simultaneouslyobservingfluorescentsignalsfromagreen-fluorescentprotein（GFP）chimeraandfromtheMitoTrackerdye24–27（seeNote12.1"ProductHighlight:

FluorescentProbesforUsewithGFP"inSection12.1）

∙StudyingthelocalizationofmitochondriainfibroblaststransformedwithcDNAofwild-typeandmutantkinesinheavychains28

∙VisualizingmitochondriawhilecharacterizingthesubcellulardistributionofcalciumchannelsubtypesinAplysiacalifornicabagcellneurons29andoftheverotoxinBsubunitinVerocells30

OurVybrantApoptosisAssayKit#11（V35116,Section15.5）utilizesMitoTrackerCMXRosincombinationwithAlexaFluor488annexinVinatwo-colorassayofapoptoticcells（Figure15.95）.MitoTrackerOrangeCMTMRosanditsreducedformCM-H2TMRoshavealsobeenusedtoinvestigatethemetabolicstateofPneumocystiscariniimitochondria.31Followingfixation,theoxidizedformsofthetetramethylrosamineandX-rosamineMitoTrackerdyescanbedetecteddirectlybyfluorescenceorindirectlywitheitheranti-tetramethylrhodamineoranti–TexasReddyeantibodies（A6397,A6399;Section7.4）.

MitoTrackerGreenFMProbe

MitochondriaincellsstainedwithnanomolarconcentrationsofourpatentedMitoTrackerGreenFMdye（M7514）exhibitbrightgreen,fluorescein-likefluorescence（Figure12.13,Figure12.33,Figure14.68,Figure16.21）.TheMitoTrackerGreenFMprobehastheaddedadvantagethatitisessentiallynonfluorescentinaqueoussolutionsandonlybecomesfluorescentonceitaccumulatesinthelipidenvironmentofmitochondria.Hence,backgroundfluorescenceisnegligible,enablingresearcherstoclearlyvisualizemitochondriainlivecellsimmediatelyfollowingadditionofthestain,withoutawashstep.

UnlikeMitoTrackerOrangeCMTMRosandMitoTrackerRedCMXRos,theMitoTrackerGreenFMprobeappearstopreferentiallyaccumulateinmitochondriaregardlessofmitochondrialmembranepotentialincertaincelltype