Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization.

0.25 MB·English

by  Smith, Graham S.T.

JournalofVisualizedExperiments www.jove.com
VideoArticle
Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized
Oligodendroglial Cells using Live-cell Imaging and Cleaveable
Fluorogenic-dye Substrates Following Potassium-induced Membrane
Depolarization
GrahamS.T. Smith,JanineA.M. Voyer-Grant,George Harauz
DepartmentofMolecularandCellularBiology,UniversityofGuelph
URL:http://www.jove.com/video/3422/
DOI:10.3791/3422
Keywords:Neuroscience,Issue59,myelinbasicprotein,apoptosis,neuroprotection,caspase-3,live-cellimaging,glia,oligodendrocytes,
DatePublished:1/13/2012
Citation:Smith, G.S.,Voyer-Grant, J.A.,Harauz, G. MonitoringCleavedCaspase-3ActivityandApoptosisofImmortalizedOligodendroglialCells
usingLive-cellImagingandCleaveableFluorogenic-dyeSubstratesFollowingPotassium-inducedMembraneDepolarization.J.Vis.Exp.(59),
e3422,DOI:10.3791/3422(2012).
Abstract
Thecentralnervoussystemcanexperienceanumberofstressesandneurologicalinsults,whichcanhavenumerousadverseeffectsthat
ultimatelyleadtoareductioninneuronalpopulationandfunction.Damagedaxonscanreleaseexcitatorymoleculesincludingpotassiumor
glutamateintotheextracellularmatrix,whichinturn,canproducefurtherinsultandinjurytothesupportingglialcellsincludingastrocytesand
oligodendrocytes8,16.Iftheinsultpersists,cellswillundergoprogrammedcelldeath(apoptosis),whichisregulatedandactivatedbyanumberof
well-establishedsignaltransductioncascades14.Apoptosisandtissuenecrosiscanoccuraftertraumaticbraininjury,cerebralischemia,and
seizures.Aclassicalexampleofapoptoticregulationisthefamilyofcysteine-dependentaspartate-directedproteases,orcaspases.Activated
proteasesincludingcaspaseshavealsobeenimplicatedincelldeathinresponsetochronicneurodegenerativediseasesincludingAlzheimer's,
Huntington's,andMultipleSclerosis4,14,3,11,7.
InthisprotocolwedescribetheuseoftheNucView488caspase-3substratetomeasuretherateofcaspase-3mediatedapoptosisin
immortalizedN19-oligodendrocyte(OLG)cellcultures15,5,followingexposuretodifferentextracellularstressessuchashighconcentrationsof
potassiumorglutamate.Theconditionally-immortalizedN19-OLGcellline(representingtheO2Aprogenitor)wasobtainedfromDr.Anthony
Campagnoni(UCLASemelInstituteforNeuroscience)15,5,andhasbeenpreviouslyusedtostudymolecularmechanismsofmyelingene
expressionandsignaltransductionleadingtoOLGdifferentiation(e.g.6,10).Wehavefoundthiscelllinetoberobustwithrespecttotransfection
withexogenousmyelinbasicprotein(MBP)constructsfusedtoeitherRFPorGFP(redorgreenfluorescentprotein)13,12.Here,theN19-OLG
cellculturesweretreatedwitheither80mMpotassiumchlorideor100mMsodiumglutamatetomimicaxonalleakageintotheextracellularmatrix
toinduceapoptosis9.Weusedabi-functionalcaspase-3substratecontainingaDEVD(Asp-Glu-Val-Asp)caspase-3recognitionsubunitanda
DNA-bindingdye2.Thesubstratequicklyentersthecytoplasmwhereitiscleavedbyintracellularcaspase-3.Thedye,NucView488isreleased
andentersthecellnucleuswhereitbindsDNAandfluorescesgreenat488nm,signalingapoptosis.UseoftheNucView488caspase-3
substrateallowsforlive-cellimaginginreal-time1,10.Inthisvideo,wealsodescribetheculturingandtransfectionofimmortalizedN19-OLGcells,
aswellaslive-cellimagingtechniques.
VideoLink
Thevideocomponentofthisarticlecanbefoundathttp://www.jove.com/video/3422/
Protocol
1. Thawing and Culturing Cells
1. ObtainstockoffrozenimmortalizedN19-oligodendroglialcellsfromlongtermliquidnitrogenstores.
2. Immersevialcontainingcellsat37°Cwaterbathuntilthecellsuspensioniscompletelythawed.
3. Add7mLofDMEM(Dulbecco'sModifiedEagleMedium)withhigh-glucosesupplementedwith10%FBS(FetalBovineSerum)and1%
penicillin/streptomycintoa10cmcultureplate.
4. Addthecell-suspensiondrop-wisetotheplate,andgentlyagitateandrockthePetriplatetodispersethecellsevenly.
5. Culturecellsat34°C/5%CO2incubator.
6. After4hours,aspiratemediafromplatestoremoveanyremainingDMSO(dimethylsulphoxide)thathadbeenusedasacryoprotectant,and
replacewithfreshmedia(7mLDMEMhigh-glucosemediasupplementedwith10%FBSand1%penicillin/streptomycin).
2. Passaging Cells
1. At70-80%confluence(4-7daysgrowth),aspiratemediafromcells.
2. Add1mLof0.25%trypsintoplate.Pipettetrypsintodetachcells(about5minutes).
3. Maketheappropriatedilutionsforexperimentalconditionsandpassageanadditional10cmplateforfutureexperimentsatacelldensityno
lessthat0.1x106cells/mL.
Copyright©2012 CreativeCommonsAttributionLicense January2012| 59 |e3422|Page1of5
JournalofVisualizedExperiments www.jove.com
Note:Youshouldpassageyourcellsatleasttwiceafterthawingbeforeusingthemforexperiments.
3. Counting and Plating Cells
1. Toplatecellsforlive-cellimaging,trypsinizeaspreviouslydescribed.
2. Removeapproximately30μLofcellsandcountusingahaemocytometer.
3. Placeanuncoatedglasscoverslipina6wellplate.Addcellstothewellatadensityof0.1x106cells/mLin2mLphenol-freeDMEM
high-glucosemedia,supplementedwith10%FBSand1%penicillin/streptomycin,at34°C/5%CO2.
4. Allowcellstogrowovernight(16-20h)at34°C/5%CO2beforetransfection.
4. Transfection
1. Combine100μLserum-freemedia,0.5-4μgpurifiedplasmidDNA,and4μLFuGENEHD(Roche).Vortexgentlytomix.
2. VortexagainbrieflyandallowtheDNAtocomplexfor5minatroomtemperature,andthenaddFuGENEHDDNAmixturedirectlytothe
culturedcells.Tilttheplategentlytomix.
3. Culturethecellsforanadditional48hat34°C/5%CO2priortotreatmentorexperimentation.
5. Preparing Cells for Live-Cell Imaging (LCI)
1. TurnontheLCIChamlidelive-cellinstrumentcontrolboxatleast1hbeforeyouwanttostartyourexperiment.Thecontrolboxalsoregulates
thetemperatureandhumiditywithintheLCIChamlide,andregulatestheflowofthepremixed5%CO2.
Note:Itisadvisabletoturnonthecontrolboxupto3hbeforebeginningexperimentstoensurethattheentirestagereaches34°C.Thisstepwill
reducefocaldrift,whichiscausedbyfluxulationandthermalexpansionofthemetalstageasitwarms,duringimaging.
2. Workinginaflow-hood,spraytheChamlidemagnetic-typeculturechamber,andtweezers,with70%ethanolandallowthemtodryfor5
minutes.
3. Removecellsfromincubatorandconfirmthattheyarehealthy.Theyshouldappearadherentandwell-spreadontheglasscoverslipwith
numerousmembraneprocesses.Cellsthatarestressedfromtransfectionarenotsuitableforexperimentation,andwillhaveareduced
numberofprocessextensions,andoftenhaveanirregularly-shapednucleus.
4. Tiltthe6-wellplateandremovethecoverslipwithtweezers.Quicklyplacethecoverslipcellsideupintothebottomplateoftheculture
chamber.Donotallowthecoversliptodry.
5. Attachthemagneticmainbodyoftheculturechamberandadd500μLofmediafromtheoriginal6-wellplateontothetopofthecoverslip.
Re-usingthismediawilldecreasetheamountofstressplacedonthecellscausedbyenvironmentalchanges,andcanalsobeusefulfor
assessingextracellularsecretedfactors.
6. Placetheglasscoverontheculturechamber.
7. UseaKimWipesprayedwith70%ethanoltoremoveanyresidualmaterialfromthebottomofthecoverslip,whichwouldinterferewith
microscopy.
8. Placetheculturechamberinthe34°C/5%CO2incubatorfor30min.Thisstepwillensurethatthechamberitselfwarmsto34°Ctoreduce
shiftingofthecoverslipasthemetalwarmsup.
6. Microscope Settings
ImageswereacquiredhereusingaLeicaDMIRE2invertedmicroscopewithacustomrelaylensandemissionfilterwheelhousingforcartridge
loadingofmultiplewheels(QuorumTechnologiesInc.,Guelph,ON).
1. Brightfield-Lampsetto2.5Vandexposuretimeto240ms.
2. RedFluorescentProtein(RFP)-Gainsetat140for200ms.
3. GreenFluorescentProtein(GFP)-Gainsetat140for500ms.
4. Ourmicroscopehasadimmablelampratherthanaspinningdisktocontroltheamountoflightavailabletocells.Werunourexperimentswith
thelightat90%ofmaximumlampintensity.
7. Treatment of Cells withApoptosis Inducers and NucView 488 Caspase-3 substrate
1. Itisimportanttopreparelargestocksofapoptosisinducersaheadoftimeandfreezetheminaliquots,sothattheconcentrationswillbe
consistentbetweenexperiments.
2. TheNucView488substrateislightsensitive.Preparealiquotsof15μLtoreducefreeze/thawing,andstoretubesat-20°Ccoveredin
aluminumfoil.WorkwiththeambientroomlightingaslowaspossibletoreduceexposureoftheNucView488substratetolight,priortoits
use.
3. Retrievetheculturechamberquicklyfromtheincubatorsothatthecellsarenotexposedtoareductionintemperature.Placetheculture
chamberontotheenvironmentalchamberofthemicroscope,anduseclampstokeeptheculturechamberfrommoving.Atthispointyou
shouldalsodimtheroomlights.
4. Turnonthe5%premixedCO2tankwithdualregulator.
5. Usingthe10xobjective,focustofindcellsonthecomputermonitorusingbright-fieldmicroscopy.Note:youwouldwanttousethelargest
numericalapertureontheobjectivewiththedesiredmagnificationtoreducetheexposuretime.
6. Deliverinducersofapoptosistothecells(inourcase80mMpotassium,or100mMglutamate),oneofthefollowingmethodscanbeused.
Wewillusedeliveryof80mMpotassiumasanexample,usingKCldissolvedasa10xconcentrateinourtypicalculturemedia.
1. Mediaexchangebyslowandlocalperfusion:

-Useaperistalticpumptoexchangemediaintheculturechamberwithnewmediacontaining80mMpotassium.

-Oneendofthetubingwilldeliver10mLofastockof80mMpotassiumdissolvedinphenol-freeDMEM,andtheotherendofthe
tubingwillremovemediafromthechamber.
Copyright©2012 CreativeCommonsAttributionLicense January2012| 59 |e3422|Page2of5
JournalofVisualizedExperiments www.jove.com

-Youwantatleasta10xexchangeofmediatoensurethatthemedialeftinthechamberhasthecorrectconcentrationofpotassium.

-Aftermediaisexchanged,add3μLofNucView488substratetothemediainthechamber.Pipettetomix.
2. Directadditionof80mMK+andNucView488substratetomediainculturechamber:

-Preparea10xstocksolutionof80mMpotassiumdissolvedinphenol-freeDMEM.

-Add3μLofNucView488substrateto50μLof10x80mMpotassium.Pipettetomix.Addto500μLphenol-freeDMEMinculture
chamber.

Note:thismethodispreferredifyouareinterestedinmaintainingorassessinggrowthorothersecretedfactorsthatmaybepresentin
theoriginalmedia.WehavefoundthattheNucView488substrateisstableincellcultureforexperimentslastingaslongas36h.
8. Live-cell Imaging
1. Clickontheredchanneltodisplaytransfectedcells.
2. Selectandsavearoundadozenframeswherethereareseveraltransfectedcells,andsavethese"X-Ystagepoints".Dependingonthe
amountofcomputermemory,youmaybelimitedtothenumberofstagepointsthatyouwillbeabletoacquire.
3. Havethemicroscopecaptureimages(inbright-field,redchannel,andgreenchannel)atthesesavedstagepointsevery6minutes.
4. Anygreenstainingthatisvisibleduringtheearlystagesoftheexperimentslikelyindicatescellsthatarealreadyundergoingapoptosis,due
usuallytotransfectionand/orenvironmentalstress.Apoptosisduetotheexperimentaltreatmentswillbedetectedatalatertimepointinthe
experiment.
9. StatisticalAnalysis
1.  

Foreachexperiment,weprogramthemicroscopetoacquiremultipleX-Ypointstogatheralargedatasetefficiently.Eachexperimentis
performedinduplicateortriplicate,anddataarecompiledfromseparateexperimentsperformedondifferentdays.Fromeachdataset,we
analyzeupto15fieldsofviewandcomparetheratioofcaspase-negativecellstocaspase-positivecells(totalnumberofcellsinfieldof
view/totalnumberofcaspase-positivecells).

2. Therecordedmeasurementsfromeachdatasetaregroupedintoalargersampleset,andarethencomparedtooneanotherusingan
ANOVAtable(p=0.05).Weshowthestandarderrorsofthemean(SEM)ofeachexperiment,andthencomparethedifferenceinmeansby
performingaTukeymeanscomparisontest(p=0.05)todeterminewhichtreatmentsaresignificantlydifferentfromeachother.
10. Representative Results
WehavedescribedanexperimenttoillustratehowtheNucView488substratecanindicateanincreasedrateofapoptosisofN19-OLGcell
culturesfollowingatreatmentwithahighextracellularpotassiumconcentration.TheN19-cellsweretransfectedwithRFP,andwereeithertreated
with3μLNucView488substrate(control),or3μLNucView488substrateand80mM[K+](treatment).Cellsweremonitoredandimageswere
acquiredovera12htimecourse,whichisadequateforstudiesinvolvingneurologicalinsults(Figure1A).Incontrolconditions,wedidnot
observesignificantamountsofapoptosiscomparedtothe80mM[K+]treatedcultures(hashedbox),whichshowedapproximately45%celldeath
after12h(Figure1B).Thebackgroundgreensignalobservedinthecontrolconditionsindicatesthecellsthatareundergoingapoptosiswithout
theadditionofextracellularpotassium.Virtuallynoneofthecellsinthecontrolexperimentexhibitapoptosisbythe12htimepoint,althoughin
othersituationstheexperimentsmayberequiredtorunlonger.Imageswereacquiredusinga10xobjective.Bar=100μm.
Copyright©2012 CreativeCommonsAttributionLicense January2012| 59 |e3422|Page3of5
JournalofVisualizedExperiments www.jove.com

Figure1.(A)A12htimecourseexperimentofN19OLGcultures48hpost-transfectionexpressingRFP-MBP(redchannel)alongwith3μLof
NucView488substrate(greenchannel).Cultureswereeithertreatedwithafinalconcentrationof80mM[K+](leftpanels),ornotreatmentasa
control(rightpanels).Imageswereacquiredat6minintervals,andsignificantactivationofcleavedcaspase-3(greensignal)canbeobservedin
culturestreatedwith80mM[K+]withinthecellnuclei(hashedbox)comparedtothecontrolexperiment.(B)Percentageofcleavedcaspase-3
cells(calculatedbydividingthetotalnumberofcellsinthefieldofviewbythetotalnumberofcaspase-positivecells).Incomparisontocontrol
conditions,weobservedaround45%celldeathfollowingK+-treatmentby12h.
sVideo1.A12htimecourseexperimentofN19-OLGcultures48hpost-transfectionexpressingRFP-MBP(redchannel)with3μLofNucView
488substrate(greenchannel),alongwithbright-fieldimagesandathree-waymergedimage,followingtreatmentwith80mM[K+].Pleaseclick
heretosee/downloadthisvideofile.
sVideo2.A12htimecourseexperimentofN19-OLGcultures48hpost-transfectionexpressingRFP-MBP(redchannel)with3μLofNucView
488substrate(greenchannel),alongwithbright-fieldimagesandathree-waymergedimage.Notreatmentwasappliedtotheculturesand
N19-OLGscanbeseenmigratingthroughthemicroscopefieldincontrasttocellculturestreatedwith80mM[K+](comparewithsVideo1).
Pleaseclickheretosee/downloadthisvideofile.
Discussion
Althoughthisisnottheonlyfluorogenicproductavailableforapoptosisdetection,thereareseveralsignificantadvantagestousingtheNucView
488substrate.Oneofthemainbenefitsistheabilitytofollowapoptosisinlivecellsinrealtime,whereasmostalternativeproductseitherrequire
celllysisorhavepoorcellpermeability.Otherbenefitsincludehighsensitivityforcaspase-3recognition,highcellpermeability,lowcytotoxicity,
andnointerferencewiththeprogressionofapoptosis.Thesubstratealsohaslowbackgroundfluorescenceuntilitiscleavedandentersthe
nucleus,whicheliminatesbackgroundfluorescence.Thecaspase-3recognitionsequencecontains3negativechargesandtheDNA-bindingdye
hasonepositivecharge2.TheDEVD-NucView488moleculethushasanetnegativecharge,whichpreventstheactivationandbindingofthe
dyetoDNAincellswherecaspaseisnotactive.
Maintainingconsistencybetweenexperimentsisrequiredtobeabletodrawmeaningfulcomparisonsbetweenreplicates.Oneofthemain
parameterstokeepconsistentiscelldensity,asitaffectstherateoftransfection.ObtaininghightransfectionratesinimmortalizedN19-OLGcell
culturesismoredifficultthanwithothercommoncelllinessuchasHeLaorHEK293,andishighlydependentondensity,inourexperience12,13.
OurbesttransfectionefficienciesforthesecellshavebeenobtainedwithFugeneHD(Roche)andarenormallyabout15%,butcanreachover
30%,dependingontheconstruct.Carefulcellcountingwillaidinobtainingconsistenttransfectionrates.Itisalsoimportanttoexposecellsfrom
differenttreatmentstothesamedegreeofenvironmentalstress,particularlywhenmeasuringratesofapoptosis.Specifically,thelengthoftime
overwhichthecellsareexposedtotransfectionreagents,ortheamountoflightexposure,areimportantenvironmentalvariablestoconsider,and
Copyright©2012 CreativeCommonsAttributionLicense January2012| 59 |e3422|Page4of5
JournalofVisualizedExperiments www.jove.com
shouldremainconstantacrossexperiments.Forourapplications,wehavefoundthatcollectingalargenumberoffields-of-viewprovidesa
sufficientlylargesamplesizetomakestatistically-significantcomparisons[ibid].
Disclosures
Theauthorshavenoconflictsofinteresttodisclose.
Acknowledgements
ThislaboratoryhasbeensupportedbytheCanadianInstitutesofHealthResearch,theNaturalSciencesandEngineeringResearchCouncilof
Canada,andtheMultipleSclerosisSocietyofCanada(MSSC).GSTSwastherecipientofaDoctoralStudentshipfromtheMSSC.Weare
gratefultoDr.JoanBoggs(HospitalforSickChildren,Toronto)formanyhelpfuldiscussionsandcommentsonthismanuscript.Wearegratefulto
BiotiumfortheirgenerousgiftofadditionalNucView488caspase-3.
References
1. Antczak,C.,Takagi,T.,Ramirez,C.N.,Radu,C.,&Djaballah,H.Live-cellimagingofcaspaseactivationforhigh-contentscreening.J.Biomol.
Screen.14,956-969(2009).
2. Cen,H.,Mao,F.,Aronchik,I.,Fuentes,R.J.,&Firestone,G.L.DEVD-NucView488:anovelclassofenzymesubstratesforreal-timedetection
ofcaspase-3activityinlivecells.FASEB.J.22,2243-2252(2008).
3. deCalignon,A.,Fox,L.M.,Pitstick,R.,Carlson,G.A.,Bacskai,B.J.,Spires-Jones,T.L.,&Hyman,B.T.Caspaseactivationprecedesand
leadstotangles.Nature.464,1201-1204(2010).
4. Eldadah,B.A.&Faden,A.I.Caspasepathways,neuronalapoptosis,andCNSinjury.J.Neurotrauma.17,811-829(2000).
5. Foster,L.M.,Phan,T.,Verity,A.N.,Bredesen,D.,&Campagnoni,A.T.Generationandanalysisofnormalandshiverertemperature-sensitive
immortalizedcelllinesexhibitingphenotypiccharacteristicsofoligodendrocytesatseveralstagesofdifferentiation.Dev.Neurosci.15,
100-109(1993).
6. Fulton,D.,Paez,P.M.,Fisher,R.,Handley,V.,Colwell,C.S.,&Campagnoni,A.T.RegulationofL-typeCa(++)currentsandprocess
morphologyinwhitematteroligodendrocyteprecursorcellsbygolli-myelinproteins.Glia.58,1292-1303(2010).
7. Hisahara,S.,Okano,H.,&Miura,M.Caspase-mediatedoligodendrocytecelldeathinthepathogenesisofautoimmunedemyelination.
Neurosci.Res.46,387-397(2003).
8. Lau,A.&Tymianski,M.Glutamatereceptors,neurotoxicityandneurodegeneration.Pflugers.Arch.460,525-542(2010).
9. Lawrence,M.S.,Ho,D.Y.,Sun,G.H.,Steinberg,G.K.,&Sapolsky,R.M.OverexpressionofBcl-2withherpessimplexvirusvectorsprotects
CNSneuronsagainstneurologicalinsultsinvitroandinvivo.J.Neurosci.16,486-496(1996).
10. Paez,P.M.,Spreuer,V.,Handley,V.,Feng,J.M.,Campagnoni,C.,&Campagnoni,A.T.Increasedexpressionofgollimyelinbasicproteins
enhancescalciuminfluxintooligodendroglialcells.J.Neurosci.27,12690-12699(2007).
11. SanchezMejia,R.O.&Friedlander,R.M.CaspasesinHuntington'sdisease.Neuroscientist.7,480-489(2001).
12. Smith,G.S.T.,DeAvila,M.,Paez,P.,Spreuer,V.,Wills,M.K.B.,Jones,N.,Boggs,J.M.,&Harauz,G.Prolinesubstitutionsandthreonine
pseudo-phosphorylationoftheSH3-ligandof18.5kDamyelinbasicproteindecreaseaffinityfortheFyn-SH3-domainandalterprocess
developmentandproteinlocalizationinoligodendrocytes.J.Neurosci.Res.(Inpress,2011).
13. Smith,G.S.T.,Paez,P.M.,Spreuer,V.,Campagnoni,C.W.,Boggs,J.M.,Campagnoni,A.T.,&Harauz,G.Classical18.5-and21.5-kDa
isoformsofmyelinbasicproteininhibitcalciuminfluxintooligodendroglialcells,incontrasttogolliisoforms.J.Neurosci.Res.89,467-480
(2011).
14. Springer,J.E.,Azbill,R.D.,&Knapp,P.E.Activationofthecaspase-3apoptoticcascadeintraumaticspinalcordinjury.Nat.Med.5,943-946
(1999).
15. Verity,A.N.,Bredesen,D.,Vonderscher,C.,Handley,V.W.,&Campagnoni,A.T.Expressionofmyelinproteingenesandothermyelin
componentsinanoligodendrocyticcelllineconditionallyimmortalizedwithatemperature-sensitiveretrovirus.J.Neurochem.60,577-587
(1993).
16. Yu,S.P.Regulationandcriticalroleofpotassiumhomeostasisinapoptosis.Prog.Neurobiol.70,363-386(2003).
Copyright©2012 CreativeCommonsAttributionLicense January2012| 59 |e3422|Page5of5