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.VisualizingMitochondriainCellsandTissuesThemorphologyofmitochondriaishighlyvariable.Individingcells,theorganellecanswitchbetweenafragmentedmorphologywithmanyovoid-shapedmitochondria,asoftenshownintextbooks,andareticuluminwhichtheorganelleisasingle,many-branchedstructure.Thecellcycle–andmetabolicstate–dependentchangesinmitochondrialmorphologyarecontrolledbyasetofproteinsthatcausefissionandfusionoftheorganellemass.Mutationsintheseproteinsarethecauseofseveralhumandiseases,indicatingtheimportanceofoverallmorphologyforcellfunctioning(seeNote12.2TechnicalFocus: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-SelectiveProbesAlthoughconventionalfluorescentstainsformitochondria,suchasrhodamine123andtetramethylrosamine,arereadilysequesteredbyfunctioningmitochondria,theyaresubsequentlywashedoutofthecellsoncethemitochondrion'smembranepotentialislost.Thischaracteristiclimitstheiruseinexperimentsinwhichcellsmustbetreatedwithaldehyde-basedfixativesorotheragentsthataffecttheenergeticstateofthemitochondria.Toovercomethislimitation,MolecularProbeshasdevelopedMitoTrackerprobes—aseriesofpatentedmitochondrion-selectivestainsthatareconcentratedbyactivemitochondriaandwellretainedduringcellfixation.16BecausetheMitoTrackerOrange,MitoTrackerRedandMitoTrackerDeepRedprobesarealsoretainedfollowingpermeabilization,thesampleretainsthefluorescentstainingpatterncharacteristicoflivecellsduringsubsequentprocessingstepsforimmunocytochemistry,insituhybridizationorelectronmicroscopy.Inaddition,MitoTrackerreagentseliminatesomeofthedifficultiesofworkingwithpathogeniccellsbecause,oncethemitochondriaarestained,thecellscanbetreatedwithfixativesbeforethesampleisanalyzed.PropertiesofMitoTrackerProbesMitoTrackerprobesarecell-permeantmitochondrion-selectivedyesthatcontainamildlythiol-reactivechloromethylmoiety.Thechloromethylgroupappearstoberesponsibleforkeepingthedyeassociatedwiththemitochondriaafterfixation.Tolabelmitochondria,cellsaresimplyincubatedinsubmicromolarconcentrationsoftheMitoTrackerprobe,whichpassivelydiffusesacrosstheplasmamembraneandaccumulatesinactivemitochondria.Oncetheirmitochondriaarelabeled,thecellscanbetreatedwithaldehyde-basedfixativestoallowfurtherprocessingofthesample;withtheexceptionofMitoTrackerGreenFM,subsequentpermeabilizationwithcoldacetonedoesnotappeartodisturbthestainingpatternoftheMitoTrackerdyes.MolecularProbesofferssevenMitoTrackerreagentsthatdifferinspectralcharacteri