| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 510/525 |
| MW | ~600 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | CellAnalysis CellApoptosis |
| Related | VitalStains NeurologicalStains BiochemicalAssays |
- • ImprovedSolubility-lesslikelytoprecipitateinaqueoussolutions
- • GreaterConvenience-streamlinedprotocoltominimizehands-ontime
- • HigherQualitySignals-improvedsignaltobackgroundratio
- • MoreRobustAssay-optimizedprobeformoreconsistentresults
AssayPrinciple
JC-10™isacationic,lipophilicdyethatcanbeusedtomonitormitochondrialmembranepotential.Whenmitochondrialmembranepotentialislow,JC-10™emitsagreenfluorescence.Conversely,whenmitochondrialmembranepotentialishigh,JC-10™willemitagreen-orangefluorescence.
| Excitation | Emission | |
| LowMembranePotential | ~510nm | ~520nm |
| HighMembranePotential | ~510nm | ~570nm |
TheshiftinemissionisaresultofJC-10™"suniqueABIlitytoreversIBLyformaggregatesasafunctionofmitochondrialmembranepotential.Atlowmembranepotentials,JC-10™accumulationinmitochondriaislowandmostoftheJC-10™willbeinthemonomericform,whichwillemitat~520nmwhenexcitedby~490nmlightsource.Athighmembranepotentials,JC-10™accumulationinmitochondriaishighandmostoftheJC-10™willbeintheaggregateform,whichwillemitof~570nmwhenexcitedby~490nmlightsource.BecauseJC-10™aggregateformationisafunctionofmembranepolarization,thefluorescenceintensityofJC-10™aggregatesreflectsthedegreetowhichamitochondrialmembraneisdepolarizedorhyperpolarized.
Whilethereareolderprobesthatcanmonitormitochondrialmembranepotential,suchasJC-1,AATBioquest"sJC-10™outperformstheminseveralkeyaspects:
- UnlikeJC-1,whichtendstoprecipitateevenatlowconcentrations(ie.1µM),ourJC-10™probeismuchmoresolubleinaqueoussolutions.Thisallowsforeasierpreparationandhandling.
- OurJC-10™protocolhasbeenstreamlinedforminimalhands-ontime.Ourproductcomespreparedin2mg/mLsolution(ie.DMSO).Simplyadd,incubateandread.
- ExtensivetestinghasshowthatourJC-10™probehasbettersignalintensitythanotherprobes(seeFigure1)andhasahighersignaltobackgroundratio.
- Ourproductandprotocolshavebeenoptimizedtoensureconsistentresultsacrossmultipleruns.
- JC-10™hasbeendesignedforcompatibilitywithfluorescencemicroplatereaders,fluorescencemicroscopesandflowcytometersusingcommonFITC,TRITCandFL1/FL2channels(seeprotocolforadditionaldetails)
AdditionalFigures
| Spectrum | AdvancedSpectrumViewer |
1.PrepareJC-10workingsolution:
1.1 EachvialofDMSOstocksolution(100µL,2mg/mL,3mM)shouldbeusedonlyonce.Anyunusedvialsshouldbestoredat<-20oC.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
1.2 Preparea1XJC-10workingsolution:Onthedayoftheexperiment,thawanaliquotoftheJC-10stocksolutiontoroomtemperature.Preparea10to30µM1XworkingsolutioninHanksand20mMHepesbuffer(HHBS)orbufferofyourchoice,pH7-8with0.02%Pluronic®F-127.Mixthemwellbyvotexing.
Note:Forsomecelllines,workingsolutionatpH8mightpreventJC-10leakage.
2.RunJC-10assaywithafluorescencemicroplatereader:
2.1 Treatcellswithtestcompoundsforadesiredperiodoftime(Forexample,Jurkatcellscanbetreatedwithcamptothecinfor4-6hours)toinduceapoptosis.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.
2.2 Add100µL/well/96-wellplateor25µL/well/384-wellplateofJC-10workingsolution(fromStep1.2)intothecellplate.
2.3 IncubatetheJC-10loadingplateina37oC,5%CO2incubatorfor15-60min.
Note:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
2.4 MonitorthefluorescencechangeatEx/Em=490/525nm(FITCchannel)and540/595nm(TRITCchannel)forratioanalysis.
Optional:RemovetheJC-10workingsolutionfromtheplate;add100µL/well/96-wellplateor25µL/well/384-wellplateofHHBSbacktothecellplatebeforeanalysis.
3.RunJC-10assaywithafluorescencemicroscopeoraflowcytometer:
3.1 Treatcellswithtestcompoundsforadesiredperiodoftime(Forexample,Jurkatcellscanbetreatedwithcamptothecinfor4-6hours)toinduceapoptosis.
3.2 Centrifugethecellstoget1-5x105cellspertube.
3.3 ResUSPendcellsin500µLofJC-10workingsolution(fromStep1.2).
3.4 Incubateatroomtemperatureorina37°C,5%CO2incubatorfor10to30min,protectedfromlight.
Note:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
3.5 MonitorthefluorescencechangeatEx/Em=490/525nmand540/595nmwithafluorescencemicroscope(usingFITCandTRITCfilters)oraflowcytometer(usingFL1andFL2channels).
Optional:RemovetheJC-10workingsolutionfromtheplate;add100µL/well/96-wellplateor25µL/well/384-wellplateofHHBSbacktothecellplatebeforeanalysisonfluorescencemicroscope.
| References&Citations |  CitationExplorer |
Below,youmayfindasmallsamplingofspecificJC-10™applications.ToinquireaboutapotentialapplicationofJC-10™,ortoconsultwithourfluorescentdyespecialists,pleasecontactusatsupport@aatbio.comor1-800-990-8053.
- High-contentassaysforhepatotoxicityusinginducedpluripotentstemcell–derivedcells.
ResearchersuseJC-10™tomonitormitochondrialdepolarizationasanindicationofhepatotoxicityandoxidativestressininducedpluripotentstemcell-derivedcells,withtheultimategoalofdesigningareliable,high-contentandimaging-basedinvitrotoxicityassay. - High-ContentHigh-ThroughputAssaysforCharacterizingtheViabilityandMorphologyofHumaniPSC-DerivedNeuronalCultures
JC-10™wasusedtostudyneuronsderivedfrominducedpluripotentstem-cells.SinceJC-10™willaccumulateinthemitochondriaofviablecells,itwasusedtodeterminecellviabilityinahigh-throughputassaycontext. - AnticancerActivityofNewSyntheticα-Methylene-δ-LactonesonTwoBreastCancerCellLines
ResearcherschoseJC-10™toinvestigatemitochondrialmembranepotentialandmembraneintegrityincellstreatedwithnaturalproducts,suchasα-Methylene-δ-Lactones,withthegoalofdevelopingnewtreatmentsforbreastcancer. - Tetrandrineprotectsmouseretinalganglioncellsfromischemicinjury.
JC-10™wasusedinflowcytometrytostudymitochondrialmembranepotential(ΔΨm)inprimaryculturedretinalganglioncells,asanextensionofthefieldofdrugdiscoveryintopreventionofischemicinjury. - Midazolaminducescellularapoptosisinhumancancercellsandinhibitstumorgrowthinxenograftmice
Inastudyofhumancancercells,JC-10™wasemployedtotrackcellularapoptosisasafunctionofmitochondrialmembranepotential,andconsequently,mitochondrialactivity.Researcherswereinterestedinthepossibleanestheticpropertiesofmidazolamforanticancerdrugdelivery. - MitochondrialproteomicswithsiRNAknockdowntorevealACAT1andMDH2inthedevelopmentofdoxorubicin-resistantuterinecancer
JC-10™wasusedbyresearchersforthepurposesofdrugdiscovery.Inparticular,researcherswereinterestedinfindingnewtreatmentsfordoxorubicin-resistantuterinecancer,usingJC-10™tomonitormitochondrialmembranepotentialandvalidatecellviabilityresults. - Coldexposurelowersenergyexpenditureatthecellularlevel
ResearchersusedJC-10™toinvestigatetherelationshipbetweentemperatureandcellularactivity.Inparticular,researcherswantedtoexploreifcoldtemperatureactsasastressoronmitochondrialmembranepotential,withregardstotheoxidativephosphorylationprocesswhichgeneratesATP. - Susceptibilityofgametesandembryosoftheeasternoyster,Crassostreavirginica,toKareniabrevisanditstoxins
JC-10™wasusedinthestudyofspermviability,fertilizationsuccesssandembryonicsurvivalofCrassostreavirginica.Specifically,JC-10™wasusedtoquantifymitochondrialmembranepotentialinspermcellsandtodeterminepossibletoxicityeffectsofalgalblooms. - CalmodulinantagoNISTsinducecellcyclearrestandapoptosisinvitroandinhibittumorgrowthinvivoinhumanmultiplemyeloma
JC-10™wasusedbyresearcherstostudycellcycleandapoptosisinhumanmultiplemyeloma.JC-10™functionedasaprobeforthedetectionofmitochondrialmembranepotentialdepolarization,whichwascrucialtothestudyofcaspaseactivatedapoptosis. - ActivationofthemitochondrialapoptoticpathwayproducesreactiveoxygenspeciesandoxidativedamageinhepatocytesthatcontributetolivertumOrigenesis
Researcherswereinterestedinthepathwaysinvolvedwithlivertumorigenesis.Tothatend,theyusedJC-10™tostudyactivationofapoptoticpathwaysrelatedtochangesinmitochondrialmembranepotential,withtheultimategoalofdiscoveringifantioxidanttherapymighthelpsuppresslivercarinogenesis.
