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In-Cell ELISA (ICE)Application Guide

Validated antibodies, dyes, and kits

for the analysis of fixed adherent

and suspension cells

TABLE OF CONTENTS

I. Introduction.........................................................

a. Features.....................................................

b. Benefits......................................................

c. DetectionMethods.............................................

d. Adherent&Non-AdherentCells...................................

e. References...................................................

II. AntibodyValidation...................................................

a. FluorescenceICC..............................................

b. Immunoprecipitation&MassSpec.................................

c. Westernblotting...............................................

d. Reproducibility................................................

III. ICEKits............................................................

a. MitoBiogenesis(regulationofproteinexpression).....................

b. PhosphoPDH(regulationofactivityandproteinmodification)............

c. CleavedPARP(activationandinductionofapoptosis)..................

IV. CustomICEKits.....................................................

a. ICE-ValidatedAntibodies.........................................

b. LabeledSecondaryAntibodies....................................

c. ICESupportPack..............................................

V. MetAbArraymultianalyteanalysis........................................

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1www.mitosciences.com

Introduction

TheIn-CellELISA(ICE)assayplatformisarapid,high-throughputtechnologytoquantifytargetproteinlevelswithin

fixedcellsgrownin96-or384-wellmicroplates.ICEisaquantitativeimmunocytochemistrytechniquethatcombines

thespecificityofWesternblottingandthethroughputandreproducibilityoftraditionalsandwichELISAs.ICEisalso

referredtoasIn-CellWestern™orfixed-cellELISA.

ThisICEApplicationGuideincludesadiscussionoftheadvantagesandvalidationoftheICEassayplatformaswell

asMitoSciences’proprietaryapplicationofsuspensioncellstoICE.Inaddition,weprovideexampledatasetsfrom

bothfocusedandbroadexperimentstodemonstratethereproducibilityandflexibilityofICE.

MitoSciencesofferscompleteICEassaykitsfordefinedapplications,dozensofICE-validatedmitochondrialand

metabolicantibodies(availableinbulkorinsampleICEPacks)aswellassupportpacksanddetectionantibodiesto

getyoustartedwithICEexperimentsofyourowndesign.

Figure 1. A schematic of infra-red and colorimetric ICE methods. Treatments and duration of treatment before cell fixing are at the user’s

discretion. IR kits and detection method require LiCor® Odyssey® or similar instrument. Colorimetric kits and detection require a standard microplate reader.

Cells are treated

Cells are fixedand permeablized

Cells are incubatedwith primary antibody

LI-COR® Odyssey®or Aerius®

ColormetricPlate reader

Development Reagentis added for colorimetric kits

Cells are incubated with secondary antibody conjugated to IRDye®

ColormetricKits

IR Kits

Cells are incubated with secondary antibody conjugated to HRP or AP

2 In-Cell ELISA (ICE) Application Guide

Key Features

•Cellsareculturedandfixeddirectlyinamicroplate.Followingfixation,cellsarebrieflypermeabilized,blocked,

andexposedtoprimaryantibody(typicallyforanovernightincubation).Secondary(detection)antibodiesare

thenappliedandthentheplatesarescanned.AstandardabsorbanceplatereaderisusedtodetectHRPsignal

(Figure1,right)andaLiCor®Odyssey®orAerius®isusedtodetectinfraredIR-dyesignal(Figure1,left).Note

thatduplexingispossiblewhenusingIR-dyedetection.

• Fixationtodataacquisitionrequires~30minutesofhands-ontime.Alternatively,fixedcellplatesarestablefor

weekstomonthsat4°Cinthepresenceofsodiumazide.

• AntibodysignalcanbenormalizedtoJanusGreenwholecellstaintoaccountforanydifferencesinseeding

densitybetweenwells.

Benefits of In-Cell ELISA (ICE) Technology

1.FLEXIBILE

a.Measurespecificproteinlevelorpost-translationmodifications(e.g.phosphorylationorcleavageevents).

b.Suitableforadherentandnon-adherentcelltypes.

c.ColorimetricorIR-dyedetectionmethods(IR-dyesaffordduplexingwithinwells).

d.Microplateformatmakesiteasyto(1)testafewanalytesacrossarangeofcultureconditions,drug

treatments,orcelllinesinparallelor(2)testmanyanalytesacrossafewconditions.

2. FAST AND INEXPENSIVE

a.Cellsarefixedandassayeddirectlyinmicroplates.Nolysatepreparationorlysatetransferrequired.

b.96-and384-wellformatsarehigh-throughputandprimedforautomation.

c.NocaptureantibodymeanslessreagentcostthansandwichELISAs.

3.SOUND SCIENCE: SPECIFIC, REPRODUCIBLE, QUANTITATIVE

a.Directfixationinmicroplatesfreezesandpreservescellsintheirbiologicallyrelevantstate.

b.Microplateformatideallysuitedforduplicateortriplicatemeasurements(CVs<10%areexpected).

c.Signalscanbenormalizedtoatotalproteinreadoutortocellnumber.

4.MITOSCIENCES SUPPORT

a.MitoSciences’antibodiesarerigorouslyvalidatedforICEapplication.

b.Optimizedassaykitsavailable.

c.Supportpacksandaspectrumofdetectionantibodiessimplifyexperimentaldesign.


Detection Methods

ICEdatacanbecollectedwithaLiCor®Odyssey®orAerius®scannerusingIRdye®-labeledsecondaryantibodies

orastandardabsorbancemicroplatereaderusingHRP-labeledsecondaryantibodies.WhenaLiCor®infrared

readerisavailable,duplexingreadoutswithinasinglewellispossibleusingIR-800andIR-680labeledsecondary

antibodies.IRdye®secondaryantibodiesandIRDye®kitsoffermuchgreaterdynamicrangeandsensitivity.

ProtocolsforIRandHRPdetectionmethodsareidenticaluptotheadditionofsecondaryantibody(seeFigure1).

MitoSciencesprovidesanti-mouseandanti-rabbitHRP-conjugatedsecondaryantibodies,alsoanti-mouseandanti-

rabbitIRdye®conjugatedsecondaryantibodies.Inaddition,MitoSciencesoffersisotype-specificanti-mouseIRdye®

conjugatedsecondaryantibodiestoallowduplexingwithtwomousemonoclonalantibodiesofdifferingisotype.All

secondaryantibodiesareincludedinICEreagentkitsorcanbepurchasedseparately.

Suitable for Adherent and Non-Adherent Cells

AdherentcellsareinherentlysuitedforICEanalysis,astheyarereadilyfixedontothesurfaceofthemicroplate

onwhichtheyarecultured.MitoScienceshasdevelopedanovelmethodtofirmlyattachsuspensioncellstoa

microplatesurfaceyieldinghighqualitydatapreviouslyseenonlywithadherentcelllines.Thisapproachhasalso

beenparticularlyusefulintheanalysisofcellsthatarelooselyadherent,forexampleapoptoticcellsthatwould

otherwisebelostduringplatewashsteps(andthusnotquantified).Formoredetailsonanalyzingdetaching/

apoptoticcells,seepage18.

References

AsamplingofliteraturereportsdescribinguseoftheICEtechniquewithdiverseanalytesandcelltypes:(1)kinetic

studiesofβ-Cateninstabilizationinmousefibroblasts,(2)myosinphosphorylationinprimarymyocytesand(3)

multiplephospo-proteinanalysisfollowingkinaseinhibitortreatmentinavarietyofimmortalizedcelllines.

1. HannoushRN.2008.KineticsofWnt-drivenbeta-cateninstabilizationbyquantitativeandtemporalimaging.

PLoS One.3(10):e3498.

2. AguilarHN.2010.QuantificationofrapidMyosinregulatorylightchainphosphorylationusinghigh-throughputIn-

CellWesternassays:comparisontoWesternimmunoblots. PLoS One.5(4):e9965.

3. ChenH.2005.Acell-basedimmunocytochemicalassayformonitoringkinasesignalingpathwaysanddrug

efficacy.Anal Biochem. 338(1):136-42.


Antibody Validation

ICEresultsprovideaccurate,quantitativemeasurementsofcellularantigenconcentrations.However,ICEdoesnot

provideinternalconfirmationofantibodybindingspecificitywitheachexperiment,unliketraditionalWesternblots

orimmunocytochemistry,whichallowconfirmationbymolecularweightorsubcellularlocalization,respectively.

Therefore,confidenceinantibodyspecificityiscriticaltoICEdatainterpretationandreliability.AllofMitoSciences’

ICE-validatedantibodieshavebeenscreenedrigorouslyforspecificitybyimmunoprecipitation/massspectrometry,

WesternblottingandbyfluorescenceimmunocytochemistryundertheconditionsusedforICE.Documentationfor

eachantibodyandkitcontainthesedata,plusrecommendedworkingconcentrations,hostspecies,antibodyisotype

andspeciescross-reactivity.Examplesofvalidationbyimmunocytochemistry,massspectrometry,andWesternblot

areshowninFigures2,3,and4,respectively.

Fluorescence Immunocytochemistry

Figure 2. Fluorescence immunocytochemistry confirms the correct cellular locations of target proteins. ICE was performed on adherent fibroblast cells using a selection of mouse and rabbit antibodies according to MitoSciences’ ICE adherent cell protocol (MS901 ICE Support Pack). For fluorescence microscopy, wells were incubated with (green) Alexa Fluor® 488 labeled secondary antibody used at a 1/1000 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue).

Mouse mAb MS775gp45 - golgi

Mouse mAb MS716ACAA1 - peroxisomes

Mouse mAb MS750 Sirtuin1 - nucleus

Mouse mAb MS503ATP synthase - mitochondria

Rabbit pAb MSR02 E1 phosphoserine 293mitochondria

Mouse mAb MS751GAPDH - cytoplasm


Figure 3. Mass spectrometry is used to confirm the identity of targets after immunoprecipitation. As an example, a group of hybridomas that secrete mouse IgG antibodies suspected against catalase were used to immunoprecipitate their targets from liver tissue extract. For each clone, a single target band of appropriate size (60 kDa) was identified (the upper 150 kDa band is IgG antibody). The band of 60 kDa band was excised from the gel, trypsinized, and analyzed by tandem mass spectrometry. The resulting data are analyzed by Matrix Sciences’ Mascot informatics system, which generates a MOWSE score based on the expected molecular weight of the trypsinized peptides. These data confirmed the target of this ICE validate antibody MS721. While a MOWSE score of 65 is considered sufficient to provide confidence in the accuracy of the identification of the target, MitoSciences’ antibodies typically score many times that number (typical MOWSE scores for MitoSciences’ antibodies are between 1000 and 3000).

Figure 4. Many ICE validated antibodies also function in Western blot so targets can be confirmed by mass dependant migration. Additionally Western blot confirms relative quantitation. In this example human cell lines deficient in various enzymes of mitochondrial fatty acid beta-oxidation (gray) are compared to a series of normal fibroblast cell lines (black). By ICE analysis (A) and Western blot (B) the patients had significantly higher levels of peroxisomal 3-ketoacyl-CoA thiolase (ACAA1) - indicative of a compensatory increase in peroxisomal fatty beta-acid oxidation. Notice that patient 482 has the highest signal in both methods but is relatively lower by Western blotting. This is likely due to saturation of enzyme catalyzed signal in Western blot, while IR fluorescent ICE has a broader dynamic range.

Western Blotting

Immunoprecipitation and Mass Spectrometry

A

B


Reproducibility

ICEisahighlyreproducibletechnique.Todemonstratethiswithinthewellsofasingle96-wellplate,tissueculture

cellsweretreatedwithChloramphenicol(CAM)ordideoxycitidine(ddC)continuouslyfor7days.CAMisanantibiotic

thatinhibitsmitochondrialproteinsynthesis,ddCisnucleosideanalogandreversetranscriptaseinhibitorwhichalso

inhibitsmitochondrialDNAreplication.BydifferentmechanismsbothreducetheexpressionofmitochondrialDNA

encodedpolypeptides,includingOXPHOSComplexIVsubunitCOX-I.Asshownbelow(Figure5),CAMtreatment

causedan80%decreaseintheexpressionofCOX-I,relativetoDMSOtreatedcells.

Coefficientsofvariation(CVs)aretypically

<10%forICEexperiments.Inthisexample,

theZfactorsforDMSOvs.backgroundand

DMSOvs.CAMare0.68and0.64,respectively,

indicatingthatthisisarobustassay.

SimilarreductionswereseeninHepG2cells

whentreatedwitheitherCAMorddCthen

analyzedonthreeseparatedays(Figure6).

Three96wellplatescontainingtreatedtissue

culturecellswereanalyzedonsubsequentdays

forinter-assayvariation.

Cellswerefixedandstoredat4°C.ICE

analysiswasperformedoneachplateon

consecutivedays-1,2,3.Measurementswere

madeinduplicateusingIRdye®detection.

ThetreatmentinducedreductioninCOX-I

signaloverthreedayswas89%±3(CAM)

and72%±2(ddC).Theinterassayvariability

oftheuntreatedsamplewas<7%acrossthe

threedays(notshown).TheICEtechnique

hasexcellentinter-platereproducibility;itis

determinedthatthedegreeofexperimental

reproducibilityisprincipallydependanton

consistenttissuecultureandcellseeding.

Figure 5. ICE intra-plate variability. HeLa cells were treated with 10µM Chloramphenicol (CAM) or Vehicle (DMSO) for 7 days and analyzed by ICE for levels of OXPHOS Complex IV (COX-I, MS404). Statistics are shown for background (no primary antibody, 16 data points) ,DMSO and CAM treatments (40 data points

each, CVs=6% and 4%, respectively) using IRdye® detection.

Figure 6. ICE inter-plate variability. HepG2 cells were treated with 10µM Chloramphenicol (CAM), 10µM dideoxycytidine (ddC) or Vehicle (DMSO) for 7 days and analyzed by ICE for levels of OXPHOS Complex IV (COX-I, MS404).

CAM ddC


ICE Kits

Overview

Screeningnewdrugcandidatesearlyinthesafetyscreeningprocessrequiresanassaythatisadequately

quantitative,specific,andreproducible,andwhichisflexibleenoughtobesuitableforavarietyofcelltypes.Itmust

alsohaveaminimumofsamplestepsand,ideally,beadaptabletoautomation.MitoSciences’ICEkitsprovide

researchersandtoxicologistswithasolutionthatmeetsalloftheserequirements.

Kit 1 - MitoBiogenesis In-Cell ELISA (MS642, MS643)

Background

MitoBiogenesisICEKitsarehigh-throughputassaysformeasuringtheratioofsteady-statelevelsofamitochondrial

DNA-andanuclearDNA-encodedproteininculturedorprimarycells.

Theadditionof“new”mitochondrialmaterialwithinacell,i.e.mitochondrialbiogenesis,requiresthecoordinated

synthesisof13proteinsencodedontheorganelle’sownDNAandmadeonmitochondrialribosomes,alongwith

thousandsofnuclearDNA-encodedsubunits,allofwhichareimportedintotheorganellebywayofnowwell-

definedtransportprocesses.MitochondrialDNAandribosomesdifferfromthechromosomalDNAandcytosolic

ribosomesinseveralaspects,reflectingtheirprokaryoticorigininevolution.Thesimilaritybetweenmitochondrial

biogenesisandbacterial/viralreplicationrepresentsasignificantchallengefordrugdevelopersdesigningnovel

antibacterialsandantivirals,asmanyofthesedrugscancauseseriousmitochondrialtoxicity.MeasuringRNAlevels

usingreversetranscriptionqPCRisoccasionallydiscussedasausefulhigh-throughputtechniqueforidentifying

effectsonmitochondrialbiogenesis,butthisapproachhasafundamentallimitation:itsinabilitytoidentifyinhibitionof

proteintranslation.IfsuchaneffectisoccuringthenRNAlevelsmightinfactincreaseinthecell,sincemitochondrial

translationisunabletoprocesstranscriptionproducts.Theonlywaytoeasilyandsimultaneouslyidentifyinhibition

ofbothmitochondrialtranscriptionandtranslationistoratiothelevelsofanmtDNAencodedproteinwithtotalcell

proteinand/orannDNA-encodedprotein.ThiscanbedonebyWesternblottingorbyICCmicroscopy,butneither

techniqueissuitablyquantitative,noraretheyamenabletohigh-throughputnorclinicalapplications.MitoSciences

offerstheonlysolutionformeasuringdrug-inducedeffectsonmitochondrialbiogenesisearlyinthesafetyscreening

process.OurMitoBiogenesisICEAssayisatrueduplexing96/384-wellassaythatratiosbothanmtDNA-andan

nDNA-encodedproteininculturedorprimarycells,andwhichrequiresverylittlesampleprepandfewoverallsteps.


Assay Principle

Cells(human,ratormouse)areseeded

in96-or384-wellmicroplates,andafter

exposuretoexperimentalcompounds

forseveralcelldoublings,thelevelsof

twomitochondrialproteinsaremeasured

simultaneouslybyspecificantibodiesin

eachwellbyICE.Thetwoproteinsare

eachsubunitsofadifferentoxidative

phosphorylationenzymecomplex,

ComplexIV(COX-I),whichismtDNA-

encoded,andthe70kDasubunitof

ComplexII(SDH-A),whichisnDNA-

encoded.AfterIRorcolorimetric

analysis,cellscanbestainedwith

thewholecellstainJanusGreenfor

thepurposesofnormalizingsignals.

Normalizinglevelsofbothproteinsto

wholecellstainingcanprovideadditional

informationregardingtheeffectsofdrugs

orconditionsonmitochondrialmassand

biogenesispercell.Drugsthatadversely

affectmitochondrialbiogenesissuchas

antibioticsoranti-retroviralnucleoside

analogsareidentifiedeasilywiththis

assay(Figure7),anddoseresponse

seriesareeasilyruntoquicklyestablish

IC50data(Figure8).

Figure 7. mtDNA-encoded protein expression is reduced specifically in rat H9C2 cardiomyocytes treated in vitro with the antibiotic chloramphenicol. A dilution series of rat cardiomyocytes is shown treated with the antibiotic chloramphenicol at 10 µM. Using MS642 the levels of mtDNA-encoded COX-I (green 800 channel), nuclear DNA-encoded SDH-A (red 700 channel) and relative ratios COX-I/SDH-A (merged) are shown in each microwell. At all cell densities a constant ratio of mtDNA encoded protein expression (COX-I) to nuclear DNA-encoded mitochondrial protein expression (SDH-A) is observed. Therefore, normalizing COX-I to SDH-A simplifies data analysis and eliminates the need to perform all tests at the same cell concentration.

Control Chloramephenicol

COX-I

SDH-A

MergeCOX-I/SDH-A

100.000 cells/well

1.000 cells/well

Figure 8. Inhibition of mitochondrial biogenesis by chloramphenicol. The IC50 of a drug’s effect on mitochondrial protein translation can be determined quickly using the MitoBiogenesis ICE (IR). In this example, cells were seeded at 3000 cells/well, allowed to grow for 3 cell doublings in a drug dilution series and then the relative amounts of COX-I, and SDH-A were measured in each well. Chloramphenicol inhibits mtDNA-encoded COX-I protein synthesis relative to nuclear DNA-encoded SDH-A protein synthesis by 50% at 3.5 µM.

Availability

Thekitincludesallnecessaryreagentsandadetailedprotocolforconductingtheassay,andversionsareavailable

forbothIR(MS642)andcolorimetricdetection(MS643).

Product Information

Cat. No. Product Name Amount Reactivity

MS642 MitoBiogenesis™In-CellELISAKit(IR) 2x96tests human,rat,mouse,bovine

MS643 MitoBiogenesis™In-CellELISAKit(Colorimetric) 2x96tests human,rat,mouse,bovine


Kit 2 – PhosphoPDH In-Cell ELISA (MSP47, MSP48)

Background

MitoSciences’PhosphoPDHICEKitsarehigh-throughputassaysformeasuringphosphorylationatallthreeE1α

regulatoryserines:Ser232,Ser293andSer300andup-ordown-regulationoftotalpyruvatedehydrogenase(PDH)

subunitE1α.Theassayisdesignedforusewithculturedadherentcellsina96/384wellmicroplateformat.

Thepyruvatedehydrogenasecomplexisthekeyregulatorysiteincellularmetabolism,inthatitlinksthecitricacid

cycleandsubsequentoxidativephosphorylationwithglycolysisandgluconeogenesis,aswellaswithbothlipid

andaminoacidmetabolism.Notsurprisingly,givenitscentralroleinmetabolism,PDHisundertightandcomplex

regulation,whichincludesregulationbyreversiblephosphorylationinresponsetotheavailabilityofglucose.In

humans,PDHactivityisinhibitedbysite-specificphosphorylationatthreesitesonthePDHE1αsubunit(Ser232,

Ser293andSer300),whichiscatalyzedbyfourdifferentpyruvatedehydrogenasekinases(PDK1-4).Eachofthe

fourkinaseshasadifferentreactivityforthesethreesites.Interestingly,phosphorylationatanyonesiteleadstothe

inhibitionofthecomplexandashifttoglycolyticmetabolismofpyruvate.Twopyruvatedehydrogenasephosphatases

(PDP1andPDP2)dephosphorylatetheE1αandactivatetheenzyme.Boththekinasesandphosphatasesare

differentiallyexpressedintissues.EachofthePDKsandPDPsisundertranscriptionalcontrolinresponsetodifferent

cellularstressevents.Inaddition,thekinasesareactivatedbyacetylCoenzymeA,NADHandATP,meanwhilethe

availabilityofpyruvateandADPleadstotheirinhibition.

TheregulationofglucosemetabolismbyPDHphosphorylationisofkeyinterestinhypoxiaandtumorcellmetabolism

andistherefore,atargetforcancertherapyaswellasothermetabolicdiseasessuchasdiabetesandobesity.

Assay Principle

Cells(human,ratormouse)areseededin96-or384-wellmicroplates,andafterexposuretotreatment,thelevelsof

phosphorylatedPDHE1αateachofthreeregulatoryserinesismeasuredwhilesimultaneouslymeasuringtotalE1α

subunit.Optionally,arelativecelldensitystainisincludedfornormalization.Anexampleofatreatmentaffectingthe

phosphorylationstateofPDHE1αisexposuretodichloroaceteate(DCA)aninhibitorofPDHkinases.Figure9shows

imagesfromaplatewhereadilutionseriesofDCAwasaddedtogrowingcells.DCAdecreasedthesignalfromeach

ofthethreephosphoserinesites,whiletotalE1αisunaffected.ThisresultisconfirmedbyWesternblottinginFigure

10usingthesameantibodies.ForWesternblottingsamplepreparation,thecellsmustbeharvestedandwashedin

DCAtomaintainthedephosphoryaltedstate.InICEsuchlimitationsdonotexist,cellsarefixedinstantaneouslyina

dephosphoryaltedstate.Finallyusingthiskit,adrugdilutionseriescaneasilybeperformedinmultiplecelltypes,as

showninFigure11,where3differentcelltypeshaveIC50responsetoDCAbutthemagnitudeoftheresponsediffers

bycelltypeandbyphosphorylationsite.


Figure 9. DCA inhibits kinases and reduces phosphorylation at all three sites while total E1α is unaffected. Cells (HepG2 shown) were seeded at 50,000 cells per well and treated with millimolar concentrations of dichloroacetate (DCA) in vehicle (1% DMSO) for 2 hours. Cells were then fixed and processed according to the protocol. To account for any cell density variability between wells the ratio of PDH E1α pSer : total E1α signal should be determined.

Figure 10. Antibody specificity demonstrated by Western Blot. HepG2 cells were treated with 5 mM dichloroacetate (DCA) to inhibit PDH kinase activity. To maintain the PDH in the maximal dephosphorylated state the cells were harvested quickly and washed in DCA containing buffers before Western blot analysis. Shown the PDH E1α and pSer293 analysis with antibodies used in this kit.

Figure 11. Kinase inhibiton by dichloracetate in HepG2, HeLa and fibroblasts (HDFn) cells results in decreased phosphoserine signal. The ratio of PDH E1α pSer232, 293 and 300 to the total E1α signal was normalized to the untreated (DMSO) sample for each concentration of DCA from 0-40 mM, showing that DCA is effective at inhibiting the PDH kinases and reducing phosphorylation at all three regulatory serine residues.

Availability

Thekitincludesallthenecessaryreagentsandadetailedprotocolforconductingtheassay.Versionsareavailable

forbothIR(MSP47)andcolorimetricdetection(MSP48).

Product Information


MSP47 PhosphoPDHIn-CellELISAKit(IR) 2x96tests human,rat,mouse,bovine

MSP48 PhosphoPDHIn-CellELISAKit(Colorimetric) 2x96tests human,rat,mouse,bovine


Kit 3 - PARP-1 (cleaved) In-Cell ELISA (MSA43)

Background

ThePARP-1(cleaved)ICEKitisahighlyspecificandhigh-throughputassayformeasuringcleavedPARP1(89kDa

fragment)inhumanadherentandsuspensioncells.

PARPisa113kDanuclearDNA-repairenzymethattransfersADP-riboseunitsfromNAD+tovarietyofnuclear

proteinsincludingtopoisomerases,histonesandPARPitself.ViapolyADPribosylation,PARP1isresponsible

forregulationofcellularhomeostasisincludingcellularrepair,transcriptionandreplicationofDNA,cytoskeletal

organizationandproteindegradation.InresponsetoDNAdamage,PARP1activityisincreaseduponbindingto

DNAstrandnicksandbreaks.ExcessiveDNAdamageleadstogenerationoflargebranchedADP-ribosepolymers

andactivationofauniquecelldeathprogram.Duringapoptosis,PARP1iscleavedbyactivatedcaspase-3between

Asp214andGly215,resultingintheformationofanN-terminal24kDafragmentcontainingmostoftheDNAbinding

domainandaC-terminal89kDafragmentcontainingthecatalyticdomain.TheproteolysisofPARPthroughthis

cleavagerenderstheenzymeinactiveandthisfurtherfacilitatesapoptoticcelldeath.Thusthepresenceof89kDa

PARPfragmentisconsideredtobeanimportantandgeneralbiomarkerofapoptosis.

Theassayisdesignedforusewithculturedadherentandsuspensioncellsina96-wellmicroplateformat.The

adherentcellsundergoingapoptosisreadilydetachfromacultureplate.Thecelldetachmentoftenleadstotheir

lossandthusunderestimationoftheproportionofapoptoticcells.Thisassaywasdevelopedtoeliminatethelossof

apoptoticcells.Inaddition,thisassayisalsoapplicableforsuspensioncells.

Assay Principle

Thisassayusesacombinationofaplatecoatingandsimplecentrifugationstepstoachieveefficientfixationofcells

totheplate.Adherentcellsareseededdirectlyintotheprovidedassayplateandexposedtoexperimentalconditions

ofinterest.Suspensioncells,afterexposuretoexperimentalconditions,aretransferredtotheassayplate.Thenthe

proteinlevelsofcleavedPARParemeasured.TheprimaryantibodyusedinthisassayreactswiththeN-terminal

endofthe89kDaPARPfragmentformedbythecaspasecleavageadjacenttoAsp214;itthusrecognizesonlythe

apoptosis-specific89kDacatalyticdomainfragment,butitdoesnotrecognizethefull-lengthPARPorthe24kDa

DNAbindingdomainfragment.Optionally,acelldensitystainisincludedfornormalization.Examplesusingboth

adherentandsuspensioncellsareshowninFigures12and13.Notethecelltype-specificresponseofcleavedPARP

toStaurosporineandhighefficiencyofcrosslinkingofapoptoticcellstotheassayplate(Figure13).


Figure 12. Time-dependence of PARP cleavage in adherent cells treated to undergo apoptosis. HeLa cells were seeded as indicated in the assay plate, allowed to attach overnight, treated with Staurosporine (STS) and fixed. The plate was analyzed by ICE to measure cleaved PARP using MSA43 kit. All steps were performed as described in MSA43 Protocol. (A) Image of the assay plate analyzed by the LI-COR® Odyssey® imaging system. (B) Quantification of cleaved PARP signal after subtraction of background signal (left panel) and normalization of cleaved PARP to cell amount measured by Janus Green (right panel). Mean and standard error of the mean (n=3) is shown. Note that the normalized cleaved PARP signal is independent of cell amount used (B, right panel).

Figure 13. Cell-line dependent induction of PARP cleavage. Adherent cell lines were seeded (HeLa and HepG2 at 50,000 per well, H196 at 150,000 per well) directly in assay plate, allowed to attach overnight and treated with 1 µM Staurosporine (STS) as indicated. Suspension cells were treated with 1 µM STS or 50 ng/mL Fas antibody as indicated and transferred (HL-60 at 300,000 per well, Jurkat at 200,000 per well) in media containing 10% serum to the assay plate. Cells were fixed and the plate was analyzed by ICE to measure the cleaved PARP using MSA43. All steps were performed as described in MSA43 Protocol. Mean and standard error of the mean (n=3) is shown. (A) Cleaved PARP normalized to cell amount measured by Janus Green cell stain. Note HepG2 cells are resistant to undergoing apoptosis under these conditions, consistent with all of our previous observations. (B) Cell amount measured by Janus Green. Note no or very small differences between Janus Green staining of treated and untreated cells indicating that the treated cells undergoing apoptosis are efficiently crosslinked to the assay plate.

Availability

Thiskit(MSA43)isavailableinIRdetectionversion.Itincludesallnecessarymaterials,reagentsandadetailed

protocolforconductingtheassay.

Product Information


MSA43 PARP-1(cleaved)In-CellELISAKit(IR) 2x96tests human

A

B


Custom ICE Kits

TakeadvantageoftheflexibilityofICEtodesignyourownexperimentsaroundyourpathway(s)ofinterest.

MitoSciencesprovidesallofthereagentsrequiredtobuildyourowncustomizedICEexperiment:

ICE-Validated Antibodies:

AcollectionofMitoSciences’ICE-validatedmousemonoclonalandrabbitprimaryantibodies.Availablein

50µgorsmalleraliquotsin3,4,or10antibodypacks.

Labeled Secondary Antibodies:

SelectfromIRDye®andHRPlabeledsecondarydetectionantibodies.

ICE Support Packs:

ReagentsandprotocolsforperformingICEassayswithadherentorsuspensioncells.ICEsupportpacks

providesufficientreagentsfor5x96well-plateassays.

ICE-Validated Antibodies

MitoSciencesoffersawideselectionofmouseandrabbitantibodiespre-validatedforperformanceinICE.Antibodies

arethoroughlytestedforthequalitiesdescribedinthevalidationsectionabove.WiththeappropriatechoiceofIRDye®

labeledsecondaryantibodies,mouseandrabbitantibodiesortwomouseantibodiesofdifferentisotypecanbeusedin

thesamewell,simultaneouslygeneratingtwoindependantmeasurements.Alternatively,antibodiescanbedetected

usingtheavailableHRPconjugatedsecondaryantibodieswithoneantibodyperwell.

Thelistofcurrentlyavailableantibodies,atthetimeofwriting,isshownonthenextpage.Mostantibodiesareavailable

onMitoSciences’websitein100µL(100µg)amounts.Additionally,allantibodiesareavailableinICEPacks.ICEPacks

arecompletelycustomizableselectionsof3,5,or10antibodiespackagedinsmalleramounts-enoughforaminimumof

50wellsforeachantibody.Inthisway,apanelofantibodiesagainstsimilartargetscanbeeasilyobtainedforpathway

analysis.TheMitoSciencesICEwebpagecontainsafiltertoolforselectingindividualantibodiesaspartofanICEPack.

Includedinthefiltercriteriaareprotein,speciesreactivity,isotype,andmetabolicpathway.

Checkwww.mitosciences.comfrequentlyaswearecontinuallyaddingtoourcollectionofICE-validatedantibodies.


Cat. # Antibody Name Pathway(s) Reactivity* Isotype Availability

MS711 2,4-dienoyl-CoAreductase(DECR1) FattyAcidOxidation(Mito) h IgG1 ICEpack/100µg

MS716 Acetyl-CoAacyltransferase,peroxisomal(ACAA1) FattyAcidOxidation(Perox) h,m,r IgG1 ICEpack/100µg

MS718 Acetyl-CoAacetyltransferase(ACAT1) Ketogenesis h IgG2a ICEpack/100µg

MS786 Aconitase(ACO2) KrebsCycle h,m,r IgG2a ICEpack/100µg

MS767 Aldehydedehydrogenase,mitochondrial(ALDH2) AlcoholMetabolsim h,m,r,b IgG2a ICEpack/100µg

MS753Alpha-ketoglutaratedehydrogenase(OGDH)subunitE2

KrebsCycle h IgG1 ICEpack/100µg

MSA09 Apoptosis-inducingfactor(AIF) Apoptosis h IgG1 ICEpack/100µg

MS503 ATPsynthasesubunitbeta OXPHOS h,m,r,b,mk IgG1 ICEpack/100µg

MS776 Bcl-2 Apoptosis h IgG1 ICEpack

MS778 BNIP3 Apoptosis h,m IgG2b ICEpack

MS722 Carnitinepalmitoyltransferase2(CPT2) FattyAcidOxidation(Mito) h,m,r,b IgG1 ICEpack/100µg

MS721 CatalaseFreeRadicalScavenging,FattyAcidOxidation(Perox)

h,r,b IgG1 ICEpack/100µg

MS101c ComplexIimmunocapture OXPHOS,Apoptosis h,m,r,b IgG2b ICEpack/100µg

MS103 ComplexIsubunitGRIM-19 OXPHOS,Apoptosis h,m,r,b IgG2b ICEpack/100µg

MS204 ComplexIIsubunit70kDaFp OXPHOS h,m,r,b IgG1 ICEpack/100µg

MS304 ComplexIIIsubunitCore2 OXPHOS h,m,r,b IgG1 ICEpack/100µg

MS404 ComplexIVsubunit1 OXPHOS h,m,r,b IgG2a ICEpack/100µg

MSA04 CyclophilinD(PPIF) PermeabilityTransitionPore h,m,r,b IgG1 ICEpack/100µg

MSA06 Cytochromec OXPHOS,Apoptosis h,m,r,b IgG2a ICEpack/100µg

MS723Delta(3,5)-delta(2,4)-dienoyl-CoAisomerase(ECH1)

FattyAcidOxidation(Perox) h,r IgG2b ICEpack/100µg

MS704 Dicarbonyl/L-xylulosereductase(DCXR) XenobioticMetabolism h IgG1 ICEpack/100µg

MS782Electrontransferflavoprotein(ETF)subunitalpha(ETFA)

FattyAcidOxidation(Mito) h,m,r IgG2b ICEpack/100µg

MS758 Epoxidehydrolase1(EPHX1) XenobioticMetabolism h IgG1 ICEpack/100µg

MS709 Fumarase(FH) KrebsCycle h,r,b IgG2a ICEpack/100µg

MS751Glyceraldehyde3-phosphatedehydrogenase(GAPDH)

Glycolysis h IgG2b ICEpack/100µg

MS725 Hydroxymethylglutaryl-CoAlyase(HMGCL) Ketogenesis h IgG2b ICEpack/100µg

MS728Hydroxysteroiddehydrogenase-likeprotein2(HSDL2)

Oxioreductase h IgG1 ICEpack/100µg

MS779 Hypoxiainductionfactor1α(Hif1α) Transcription h IgG1 ICEpack

MS783 Malatedehydrogenase,mitochondrial(MDH2)KrebsCycle,Gluconeogenesis

h,m,r,p IgG1 ICEpack/100µg

MS726 Medium-chainacyl-CoAdehydrogenase(MCAD) FattyAcidOxidation(Mito) h,m,r,b IgG1 ICEpack/100µg

MS765 MicrosomalglutathioneS-transferase3(MGST3) Peroxidase h,m,r IgG2a ICEpack/100µg

MS732 Mitochondrialsuperoxidedismutase2(SOD2) FreeRadicalScavenging h,m,r,b IgG1 ICEpack/100µg

MS702Mitochondrialtrifunctionalprotein(TFP)subunitalpha(HADHA)

FattyAcidOxidation(Mito) h IgG2b ICEpack/100µg

MS733Mitochondrialtrifunctionalprotein(TFP)subunitbeta(HADHB)

FattyAcidOxidation(Mito) h IgG1 ICEpack/100µg

MSM02 Mitofilin(MF) MitochondrialMorphology h IgG1 ICEpack/100µg

MS750 NAD-dependentdeacetylasesirtuin1(SIRT1) NAD+NADHCycling h,m,r IgG1 ICEpack/100µg

MS701 Nicotinamidenucleotidetranshydrogenase(NNT)NAD+NADHCycling,OXPHOS

h,m,r,b IgG1 ICEpack/100µg

MS703 Nitrotyrosine Oxidativestress h,m,r,b IgG2b ICEpack/100µg

*Reactivity: h = human, m = mouse, r = rat, b = bovine, mk = monkey, hr=hamster, p = pig

ICE Validated MOUSE Monoclonal Antibodies


Cat. # Antibody Name Pathway(s) Reactivity* Isotype Availability

MS777Poly[ADP-ribose]polymerase1(PARP-1)(cleaved)

Apoptosis h IgG1 ICEpack/100µg

MS774 Pyruvatecarboxylase,mitochondrial(PC) Gluconeogenesis h,m,r,b IgG1 ICEpack/100µg

MSP03 Pyruvatedehydrogenase(PDH)subunitE1α Glycolysis,KrebsCycle h,m,r,b IgG1 ICEpack/100µg

MSP05 Pyruvatedehydrogenase(PDH)subunitE2 Glycolysis,KrebsCycle h,b IgG1 ICEpack/100µg

MSP49 Pyruvatedehydrogenasekinaseisoform1(PDK1) Glycolysis,KrebsCycle h,m,r,b IgG1 ICEpack/100µg

MS766 Quinoneoxidoreductase(CRYZ) XenobioticMetabolism h IgG2a ICEpack/100µg

MS773 Serine-pyruvateaminotransferase(AGXT) XenobioticMetabolism h,r,b IgG1 ICEpack/100µg

MS706Short-chain3-hydroxyacyl-CoAdehydrogenase(SCHAD)

FattyAcidOxidation(Mito) h IgG1 ICEpack/100µg

MS764 TransitionalendoplasmicreticulumATPase(VCP) Transport h,m,r IgG1 ICEpack/100µg

ICE Validated RABBIT Antibodies

Cat. # Name Pathway Reactivity* Availability

MSR14 α-Tubulin Microtubules h,m,r,b,mk ICEpack

MSR08 Acetyl-CoAcarboxylase1(ACC1) FattyAcidSynthesis h,m,r ICEpack

MSR12 Acetyl-CoAcarboxylase1(ACC1) FattyAcidSynthesis h,m,r,hr ICEpack

MSR13 Acetyl-CoAcarboxylase2(ACC2)Phospho(pSer221) FattyAcidSynthesis h,m,r ICEpack

MSR24 Bak Apoptosis h ICEpack

MSR25 Bax Apoptosis h,mk ICEpack

MSR26 Bcl-X Apoptosis h,m,r ICEpack

MSR27 Caspase3(cleaved) Apoptosis h,m,r,mk ICEpack

MSR19 ComplexIassemblyfactorNDUFAF1 OXPHOS h,m,r ICEpack

MSR09 Fattyacidsynthase(FAS) FattyAcidSynthesis h,m,r,b ICEpack

MSR11 Glucose6phosphatedehydrogenase(G6PD) Glycolysis,PentosePhosphate h,m,r ICEpack

MSR05 Glucosetransporter1(GLUT1) Glycolysis h,m,r ICEpack

MSR06 Glucosetransporter4(GLUT4) Glycolysis h,m,r ICEpack

MSR10 HexokinaseII(HK2) Glycolysis h,m,r,mk ICEpack

MSR04 Hypoxiainductionfactor1α(Hif1α) Transcription h,r ICEpack

MSR23 Lactatedehydrogenase(LDH) Glycolysis h,m,r ICEpack

MSR28 Mcl-1 Apoptosis h ICEpack

MSR18 Parkin ProteinDegradation h,m,r ICEpack

MSR07 Phosphoglyceratekinase(PGK1) Glycolysis h ICEpack

MSR17 ProhibitinChaperone/MitochondrialMorphology

h,m,r ICEpack

MSR29 Puma Apoptosis h ICEpack

MSR01 PyruvatedehydrogenaseE1αPSer232 Glycolysis,KrebsCycle h,m,r ICEpack



MSR15 PyruvatekinaseisoformsM1/M2(PKM1/PKM2) Glycolysis h,m,r,mk ICEpack

MSR16 PyruvatekinaseisoformM2(PKM2) Glycolysis h,m,r,mk ICEpack

MSR21 Sirtuin2(SIRT2) NAD+NADHCycling h ICEpack

MSR20 Superoxidedismutase(SOD1) FreeRadicalScavenging h,r ICEpack

*Reactivity: h = human, m = mouse, r = rat, b = bovine, mk = monkey, hr=hamster, p = pig

ICE Validated MOUSE Monoclonal Antibodies (cont.)


Labeled Secondary Antibodies for ICE

MitosciencesoffersawideselectionofIRDye®labeledsecondaryantibodies.IRmeasurementofferssuperior

sensitivityandgreaterdynamicrangethancolorimetricmethods.IRDyes®requireaLiCor®Odyssey®orAerius®,

arereadinthe700or800channel,andcanthereforebeduplexed.ForthepurposesofICEthisallowsthe

simultaneousin-wellanalysisoftwoanalytesdetectedbytwoantibodieswhicharedifferentiatedbyhostspecies,

orisotypeandcanthereforebedistinguishedbyacarefullychosenpairofsecondarydetectionreagents.Forusers

withoutanIRimagingsystem,colorimetricdetectionusingHRPconjugatedantibodiesinastandardabsorbance

microplatereaderisrecommendedusingspeciesspecificHRPconjugatedsecondaryantibodies.AfterIRor

colorimetricanalysis,cellscanbestainedwiththewholecellstainJanusGreenforthepurposesofnormalizing

signals.

AselectionofIRDye®labeledsecondaryantibodiesallowduplexdetectionofmouseandrabbitantibodiesormouse

antibodiesofdifferentisotypesinanIRImager.

HRPlabeledsecondaryantibodiesfordetectionofmouseorrabbitprimaryantibodies.

JanusGreenwholecellstainisincludedinICEkitsandsupportpacksbutisalsoavailableseparately.

Cat. No. Antibody Name Reactive Against Amount # ICE Plates

MS923 Goatanti-mouseIRDye®800CW allsubclasses 50µL 12

MS924 Goatanti-rabbitIRDye®680LT allsubclasses 50µL 12

MS925 Goatanti-mouseIRDye®680LT anti-IgG1 50µL 12

MS926 Goatanti-mouseIRDye®800CW anti-IgG2a 50µL 12

MS927 Goatanti-mouseIRDye®800CW anti-IgG2b 50µL 12

Cat. No. Antibody Name Reactive Against Amount # ICE Plates

MS928 Goatanti-mouseHRP,IgG(H+L) allsubclasses 50µL 12

MS929 Goatanti-rabbitHRP,IgG(H+L) allsubclasses 50µL 12

Cat. No. Antibody Name Amount # ICE Plates

MS930 JanusGreencellnormalizationstain 11mL 2


ICE Support Pack

TheIn-CellELISA(ICE)SupportPackisforusewith

suspension,apoptotic/detachingcellsandadherentcell

lines.Asoutlinedintheintroduction,ICErequiresthatthe

assayedcellsarefixedontothesurfaceofamicroplate

inwhichtheassayisperformed.Thepackcontainsfive

96-wellmicroplates,buffersandprotocolstoperform

ICEwithMitoSciencesICE-validatedantibodies.Two

protocolsareavailablewhenusingthiskit.Protocol1is

forusewithsuspensioncellsandcellslikelytodetach

underexperimentalconditions(forexample,adherentcells

undergoingapoptosisreadilydetachfromacultureplate).

Protocol2isavailableforusewithnormaladherentcells.

TheseprotocolscanbeusedwithanyofMitoSciences’

ICE-validatedantibodies.

Adherentcellsaretypicallyfixedinthesameplateinwhichthecellsaregrownunderdesiredexperimental

conditions.Thus,dependingonthetypeofadherentcells,anappropriatemicroplatesurface,thatpromotesoptimal

cellgrowthandattachment,shouldbechosen.Theabilityofadherentcellstoattachtoamicroplatemakesthese

cellsnaturallysuitablefortheICEanalysis.

Thesuspensioncellprotocolreliesoncombinationofacoatingoftheassayplateandsimplecentrifugationsteps.

Thesuspensioncells,treatedasdesired,aretransferredintotheprovidedassayplate,sedimentedbycentrifugation

and,aftertheadditionoffixative,sedimentedagain.Whenthisprotocolisused,nearlyallsuspensioncellsare

crosslinkedtotheassayplate(seeFigure14).Crosslinkedcellsremainattachedontheplatewithinthecourseof

ICEassay(seeFigure15).Inaddition,itisadvantageousthatnoserum-washingstepisrequired;thesuspension

cellscanbefixedontotheassayplatedirectlyinculturemediumcontainingserum(seeFigures14and15).Several

otherproductsonthemarketusefilter-basedmicroplatestocapturethesuspensioncells.However,theseproducts

havesomelimitations,mainly:(1)verylow-ornoserum-containingmediumshouldbeusedtoavoidcloggingthe

filterplateand(2)theICEdetectionislimitedtoendpointcolorimetricmeasurements.

ForICEassaysusingeithersuspensioncellsoradherentcells,MitoSciencesoffersanICESupportPack(MS922)

thatincludesassayplates,reagentsandprotocoltoperformtheassaywithMitoSciencesICE-validatedantibodies

(availableforpurchaseseparately).

Figure 14. Suspension cells are efficiently crosslinked to the assay plate. Untreated (CON) or 4 hour Staurosporine-treated (STS, 1 µM) Jurkat cells were seeded at 200,000 cells per well into the assay plate in media containing 10% bovine fetal serum (10F Medium) or in PBS, and fixed as described in the MS922 protocol. After the fixation the number of cells attached (fixed) to the plate was determined and expressed as percentage of total seeded cells. Mean and standard error of the mean (n=2) is shown. Note that virtually all cells (untreated or STS-treated) attached to the plate whether the fixative was added to cells in 10F media or PBS.


Cat. No. Product Name Amount

MS921 In-CellELISA(ICE)SupportPack(does not include culture treated imaging plates.) 5x96tests

MS922 In-CellELISA(ICE)SupportPack(Includes 5x tissue-culture treated 96-well black/clear imaging plates.) 5x96tests

Cells that may become detached – e.g. Apoptosis

Manyexperimentalconditionscausedetachmentofadherentcells.Thesedetachedcellsareoftenlost(andthus

unaccountedforintheanalysis)duringfixationandwashingstepsofaregularICEprotocol.Forexample,adherent

cellsundergoingapoptosisreadilydetachfromacultureplate.Thedetachmentofapoptoticcellsoftenleadsto

theirlossandthusunderestimationtheproportionofapoptoticcells.MitoScienceshasdevelopedaprotocolthat

eliminatesthelossoftheapoptotic/detachingcellsandthusthisprotocolisrecommendedforICEonadherentcells

undergoingapoptosis/detachment(seeFigure13,PARP-1(cleaved)ICEKitMSA43).Asforthesuspensioncells,

thisprotocolreliesonthecombinationofacoatedassayplateandsimplecentrifugationsteps.Theadherentcells

areseededdirectlyintotheassayplate,allowedtoattachandtreatedasdesired.Thetreatedcellsaresedimented

bycentrifugationand,aftertheadditionoffixative,sedimentedagain.Therefore,whenperformingandICEassayon

adherentcellsandcelldetachmentisofconcern,werecommendtousetheICESupportPackforsuspensioncells

(MS922)whichincludesassayplates,reagentsandaprotocolwhichcontainsspecificdetailsforefficientcrosslinking

ofdetachingcellstotheassayplate.

Availability

TheICESupportPackisavailableforadherentcellsandsuspension(ordetaching)cells(MS922).

Product Information

Figure 15. Suspension cells remain firmly attached to the assay plate within the ICE assay. Untreated (CON) or 4 hour Staurosporine-treated (STS, 1 µM) HL-60 cells were seeded in the indicated amounts into the assay plate in media containing 10% bovine fetal serum (10F Medium), media without serum (0F Medium) or PBS, and fixed as described in the MS922 protocol. The cell amount attached to the assay plate was determined by Janus green staining either just after the fixation (Panel A) or at the end of ICE assay (Panel B). Mean and standard error of the mean (n=2) is shown. Note: (1) that virtually all fixed cells remained attached to the plate within the duration of the ICE assay (compare the cell amounts in Panel A to the cell amounts in Panel B), (2) that the STS-treated cells attached nearly as efficiently as the untreated cells and (3) that the cells attach efficiently even in media containing 10% serum.


MetAbArray

MitoScienceshasappliedtheflexibility,reproducibilityandthroughputofICEtocreatetheMetAbArray,aversatile

tooltomonitoranincreasinglycomprehensivepanelofmetabolicanalytesinasingleexperiment.TheMetAbArray

leveragesMitoSciences’collectionofICE-validatedantibodiestoenableresearcherstotrackmodulationofmultiple

analytessimultaneously(makingintra-andinter-pathwayanalysispossible).Bymonitoringabroaderarrayof

analytes(includingantibodiesforproteinlevel,phosphorylationandcleavageevents,etc),theICEMetAbArray

enablesnewdiscoveriesandhypotheseswithouttheneedforexpensiveandspecializedtechnologies(e.g.mass

spectroscopy).

Sample Applications of the MetAbArray:

1.Monitoranalytechangesfollowingalterationsincultureconditions,e.g.

a.Alteringenergygeneration:glucosevs.galactosecultures

b.Metabolicreprogramminginresponsetohypoxia

2.Assessdifferencesbetweendefinedsamples,e.g.

a.Clinicalpatientfibroblasts

b.Wile-typevs.knock-outcellsforproteinofinterest

c.Intrinsicdifferencesbetweencelllines

3.Discovereffectsofpathwaypertubationsorchemicalinhibition,e.g.

a.siRNAoroverexpressionofpathwaycomponents

b.Effect(s)ofspecificmetabolicinhibitor(s)orapoptoticinducer(s)

c.Off-targetordownstreammetaboliceffectsofyourcompoundofinterest

TheMetAbArrayisinherentlyflexiblesinceanalyteselectionisatthediscretionoftheresearcher.Project-specific

MetAbArraypanelscanbeassembledfromMitoSciences’listofICE-validatedantibodies(seepages15-17).Sample

pathway-specificpanelsarelistedbelow.Visitwww.mitosciences.comtoassemblepanelICEpacksofyourown

design.

Example Analyte Panels:


Alternatively,ifabroaderscreenisdesired,contacttheContractResearchdivisionatMitoSciencesforinformationon

ourcustom64-plexMetAbArray.Grow,treat,andfixyoursamplesandsendtheplatestous;weruntheMetAbArray

andsendyoutheresults.

Example MetAbArray Studies:

Thefollowingaresampledatasetsgeneratingusingthe96-wellplateMetAbArray.Thesestudieshighlightthe

flexibilityoftheICEplatform.

I. Broad analyte comparison of two culture conditions: Glucose vs. Galactose.

Figure 16. Relative analyte levels between HepG2 cells cultured in Glucose and Galactose. Long-term cultures of HepG2 cells grown in media containing 5mM glucose or 5mM galactose + glutamine were compared using the ICE MetAbArray. Plotted here on a log2 scale are the mean intensities (triplicate measurements) each analyte from the glucose culture divided by the galactose culture. Thus, all analytes with values >1 (green bars) are present at a greater amount in glucose cultures than in galactose cultures and vice versa for values <1 (red bars). Analytes are grouped by functional pathway.


II. Comparison of apoptotic readouts across sensitive and insensitive cell lines.

Figure 17. cleaved-PARP, cleaved-Caspase3 and GAPDH were compared across HL60, HeLa and HepG2 cell lines, +/- Staurosporin treatment. HL60 (suspension) and HeLa and HepG2 (adherent) cell lines were treated +/- 1µM Staurosporine for 4 hours and analyzed by ICE for levels of cleaved-Caspase3 and cleaved-PARP (apoptotic readouts) and GAPDH (a cell normalizing control). All measurements in triplicate. Note that Staurosporine does not induce cleavage of Caspase3 or PARP in HepG2 cells. Stability of GAPDH signal +/- Staurosporin treatment indicates that apoptotic cells are not being lost from the plate. Finally, note that the absolute lack of cleaved-PARP in unstimulated cells can cause the signal to drop below background following background subtraction as in the HepG2 sample.

Figure 18. ICE analysis of hypoxic response in HeLa cells following treatment with iron chelator deferoxamine (DFO). Comparison of three distinct HIF1A (hypoxia inducible factor alpha) antibodies (#1-3) and HIF1A regulated targets BNIP3 (involved in mitochondrial autophagy) and GLUT1 (a glucose transporter). All measurements in duplicate. DFO blocks the degradation of HIF1A protein, a process that requires iron cofactor. Note that the three HIF1A antibodies show increase levels in HIF1A following DFO exposure but the fold increase is dependent on the binding properties of the individual antibodies. Similarly, BNIP3 and GLUT1 show increased levels following DFO treatment.

III. Comparison of hypoxic response within a cell line.

LI-COR®, Odyssey®, Aerius® and IRDye® are registered trademarks or trademarks of LI-COR Biosciences Inc.

Copyright © 2011 MitoSciences Inc. All rights reserved.

1850 Millrace Drive, Suite 3AEugene, Oregon 97403phone: 541.284.1800

fax: [email protected]

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