acella-tox v1 3.1 protocol · 2020-02-14 · time-point and kinetics methods the generation of atp...

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aCella–TOX™

BioluminescenceCytotoxicityassay

U.S.Patent#:6,811,990(2004)ATTENTION:AddedCDCandStopReactioninJanuary2012.FAQ’saddedinOctober2010.PROTOCOLVERSION1.3–NKcellprotocoladdedinFeb2009.Pleasecontacttechsupport@celltechnology.comorcallusat650-960-2170withanyquestions.Acknowledgements:WewouldliketothankCambridgeAntibodyTechnology(http://www.cambridgeantibody.com/),MilsteinBldg,GrantaPark,Cambridge,UK,fortheircontinuedsupportandassistanceinoptimizingourpublishedADCCprotocol.ContactInformation:Address CellTechnologyInc 48820KatoRoad Suite400B FremontCA94538 USATelephone 650-960-2170Fax 650-960-0367TollFree 8887ASSAYS(727-7297)GeneralInformation [email protected] [email protected] [email protected] www.celltechnology.com



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TableofContents

I:Introduction................................................................................................................................................3II:AssayPrinciple............................................................................................................................................3III:MeasurementModes................................................................................................................................3IV:Procedures................................................................................................................................................5

4.1KitContents:...................................................................................................................................54.2EquipmentRequired:......................................................................................................................54.3MaterialsandReagentsRequiredbutnotsupplied:.....................................................................54.4SchematicRepresentationoftheaCella-TOXassay:......................................................................64.5Reagentsetup:...............................................................................................................................6

V.Assays.........................................................................................................................................................9

5.1Targetcelltitration:........................................................................................................................95.2DeterminationofoptimalE:Tratio:..............................................................................................10

VI.AntibodyDependentCellularCytotoxicity(ADCC)PROCEDURE.............................................................12

6.1SchematicRepresentationofanADCCAssay:..............................................................................126.2FortargetcellsinSuspension:......................................................................................................126.3ADCCwithAdherentTargetCellLines..........................................................................................17

VII.CellMediatedCytotoxicity(CMC)Procedure........................................................................................21

7.1SchematicRepresentationofaCMCAssay..................................................................................217.2CMCAssayDescription.................................................................................................................21

VIII.ComplementDependentCytotoxicity(CDC).........................................................................................26IX.DrugInducedCytotoxicity.......................................................................................................................28X.STOPREACTIONFORaCella-TOX:(ForHigh-Throughputapplications)...................................................30XI.References:..............................................................................................................................................31XII.FrequentlyAskedQuestions(F.A.Q.).....................................................................................................32



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I:IntroductionCell Technology introduces aCella-TOX, a new and highly sensitive assay that employs our recentlypatentedCoupledLuminescentTechnologyforthedetectionofcytotoxicity.Thisassaycanalsobeusedto detect cytotoxicity in primary cells. The principle of the assay is quantitative measurement of thereleaseofGlyceraldehyde-3-PhosphateDehydrogenase (GAPDH) frommammaliancell linesorbacterialcells(1,2,3,4).Thisenzymeisabundantlypresentinallknownlivingcells.Otherenzymereleaseassays,suchas the Lactate Dehydrogenase (LDH) release assay (5,6,7,8), suffer from low sensitivity as a result ofinterferencebyserumorphenolredpresentinthemedia.aCella-TOXcanworkinthepresenceofbothofthesemediaconstituentsandallowsovernightassayprotocolswhileretainingsensitivity.II:AssayPrincipleGAPDH is an important enzyme in the glycolysis pathway. This homotetrameric enzyme catalyzes theoxidativephosphorylationofD-glyceraldehyde-3-phosphateto1,3-diphosphoglycerate.IntheaCella-TOXscheme,thereleaseofGAPDHfromdyingcells leadstoATPproduction,whichisthencoupledwiththeluciferase/luciferinBioluminescencemethodologyproducinglight.III:MeasurementModesCytotoxicity. aCella-TOXmaybeaddeddirectly toeitheracell cultureor supernatant todetectenzymereleased from cells that have lostmembrane integrity. It is not necessary to remove live cells prior tomeasurement.Noformofpre-treatmentisneeded.Theluminescentreadoutbeginstoriseimmediatelyandmaybereadasearlyasa fewsecondsafteradditionof thereagent,orupto2hours later. In thismode,theassayisnon-destructiveandcontinuous,allowingmonitoringofadditionalparameterssuchasgeneexpression.Note: When setting up cellular mediated cytotoxicity assays, it is important to include controls todistinguishfromspontaneous(1)effectorscelldeathvs.(2)effectorcellmediatedtargetcelldeath.Thiscanbeaccomplishedbyincludingcontrolwellsofeffectorcellsaloneattheirvariouseffector:targetcellratios to measure their spontaneous cell death. Target cell death is represented by the differencebetweenthetwomeasurements(2-1).Cytotoxicity/Proliferation (Dual Mode). aCella-TOX may be used to measure both cytotoxicity and cellviability(orproliferation).Cellsaregrownin200µLofmedia.

1. Forcellviability:spindownplateandpipette100µLofcellfreesupernatantinawhiteopaque96wellplate.AddaCella-TOXreagentsasdescribedintheprotocol.

2. For cellproliferation: to the remaining100µLof cells add the lytic reagentasdescribedbelowandproceedwiththeaCella-TOXreaction.Viabilityisrepresentedbythedifferencebetweenthetwomeasurements.

Time-Point and Kinetics Methods The generation of ATP being an ongoing process, readings can bemeasured at specified time intervals and the kinetics of the reaction plotted. A 1:1 ratio of sample(supernatant)andEnzymeAssayReagent(seebelow)shouldbemaintainedinordertomaximizesignaltonoiseratios.



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AssayPrinciple

Healthy Cell

Dead or Dying Cell

Release of GAPDH Enzyme

GAPDH Enzyme

Coupled Luminescent Technology

ATP

Luciferase + Luciferin

Light



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IV:Procedures4.1KitContents:Catalog#-CLATOX100-3(500tests):ComponentDescription Part# Volume Storage1.Component1:4XEnzymeAssayReagent 6001 26ml -20oCto-40°C2.Component2:1XEnzymeAssayDiluent 3008 60ml 2-8oC3.Component3:Glyceraldehyde3-Phosphate(G3P)***checklabelforconc.

6003 »0.20ml -20oCto-40°C

4.Component4:50XDetectionReagent(Luciferase/Luciferin)

6002 0.55ml -20oCto-40°C

5.Component5:5.5XDetectionAssayDiluent 3009 5.5ml -20oCto-40°C6.Component6:LysisBuffer 3035 5.0ml 2-8oC

CLATOX100-4(1000tests)issuppliedas2XCLATOX100-3kits.CLATOX100-3L:5x96wellwhiteluminescenceplatesincludedwiththeabove500testkit.CLATOX100-3P:5x96wellwhiteluminescenceplatesincluded+5x96wellTissueCultureplatesalsoincludedwiththeabove500testkit.4.2EquipmentRequired:96wellplateLuminometer.Centrifugewithmicroplaterotorattachment.4.3MaterialsandReagentsRequiredbutnotsupplied:

1.OpaqueWhite96wellplatesforluminometer*.(PerkinElmerOptiplates#6005290orCorning#3605withcover,#3099)

IMPORTANT:Pleaserefertoluminometerguideforappropriateplatespecifications.2.Leukopack,LRSchamberorBuffypack.(sourceoffreshblood)3. ADCCCulturemedia(RPMI1640supplementedwith10%lowIgGFBS,GIBCO#16250,NEAAand

PSG).4. Histopaque,SigmaCat.#1077orFicoll-PaquePlusfromGEHealthcare,Cat.#17-1440-025. Sterile-filteredsolutionsof0.2%NaCland1.6%NaClforRBCLysis.6. Sterilefilteredsolutionof1XPBSwith2mMEDTA7. MiltenyiBiotecMidiMACSStartingkit,(Cat.#130-042-301)includingMidiMACSseparationunit,

1MACSMultistand,25LScolumnsandCD56Microbeads(Cat.#130-050-401).Pre-separationfilters(Cat.#130-041-407LS)arenotsupplied,butmayberequiredifcellpelletisclumpy.

8. TargetcellssuchasDaudi,Raji,RamosorbreastcancercelllinessuchasSKBR3orMCF7etc.9. AnantibodysuchasCommercialRituximab(humanizedAntiCD20),Herceptin(antihumanized

HER2/neu)oranothertestantibodyforADCC.10. TrypanBlue11. 96wellU-bottom,steriletissuecultureplateforADCCorCMCreaction.(TPPCat.#TP92097

availablefromMid-Sci)



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4.4SchematicRepresentationoftheaCella-TOXassay:50µLofreactionsupernatantfromADCCorCMCreaction +50µLofComponent2(Part#3008:EnzymeAssayDiluent) 100µLof2XEnzymeAssayReagent(2XComponent1:Part#6001+Component3:Part#6003:G3P) 50µLof1XDetectionReagent(1XComponent5:Part#3009+Component4:Part#6002:50XDetectionreagent) Readonluminometer.4.5Reagentsetup:1.The4XEnzymeAssayReagent:(Component1):Part#6001(Greenlabel)For100tests(one96wellplate),10mlofthe2Xreagentisrequired.Component1isafrozen4Xliquidconcentrate.Tothawthecontents,thevialshouldbeplacedinalukewarmwaterbath,orallowedtoequilibratetoroomtemperaturebyleavingonthebenchoroniceforabouthalfhour.DONOTthawinincubatororsubjectcontentstohightemperatures.Uponthawing,immediatelyaliquotandfreezetherestbetween-200Cand-40°Cinpolypropylenetubes;ifyouarenotusingthecompletecontentsofthevial.(Shorttermstorage:Ice)10mlofthe2Xcocktailisrequiredfor100tests(one96wellplate).Tomakea2Xcocktail,dilutethe4Xconcentrate1:1withtheEnzymeAssayDiluent(Component2).Forexample,to5mLof4Xenzymeassayreagent,add5mLofAssayDiluentandaddG3P(Part#6003;Component3)justpriortouse.Fordilution,seestep2.CautionaryNotes:Avoidrepeatedfreezethawcycles.If5platesofexperimentsareplanned,dilutetheentirecontentsofPart#60011:1withPart#3008.Make5aliquotsof10mleachfor500reactions.Storealiquotsat4°C.Bringeachaliquottoroomtemperatureabout10minutespriortoaddition.TheG3P(Part#6003)shouldbeaddedtothe2XsolutionIMMEDIATELYbeforethereaction.Preparationof2XEnzymeAssayReagent:For250reactionson4plates,13mlof4XEnzymeAssayReagent(Component1:Part#6001)

+13mlofEnzymeAssayDiluent(Component2:Part#3008)makes26mlof2XEnzymeAssayReagent Aliquot6mlpertubeandstoreat4°Coronice Equilibrateeachtubefor10minutesatRT Add21µLG3P(Component3:Part#6003)justbeforeaddingtoreactionwells.



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2.Glyceraldehyde3-phosphate(Component3):Part#6003(Greenlabel)NextquicklythawComponent3(G3P).Checkviallabelforconcentrationinmg/ml.

KITLOT# G3Pconcentrationonviallabelinmg/ml

VolumeinµLtobeaddedpermLof2Xcocktail

45 3.9KN21110 46 3.8

KN10306,KN20406,KN20606,KN50507,KN20112

47 3.7

48 3.6 49 3.6

KN40906,KN20906,KN40108,KN30708,KN21008,KN20409,KN20909,KN20210,KN30210

50 3.5

51 3.4 52 3.4 53 3.3 54 3.2 55 3.2

AliquotG3Pintosingleusepolypropylenevials,andstorebetween-20°Cand-40°C.Use the above table to determine the volume of G3P to be added to the 2X Enzyme Assay Reagentpreparedabove.Forexample,ifG3Pconcentrationonvialis50mg/ml,add3.5µLofComponent3(G3P)toeachmLof2XEnzymeAssayReagent(fromstep1above).Therefore,to10mlof2XReagentprepared,add35 µLofG3Ptomakesufficientcocktailfor100reactions.PreparesufficientamountofthisEnzymeAssayReagentforafulldayofuse(shorttermstorage:onice).Add100µLofthisEnzymeReactionCocktailcontainingG3Ptoevery100µLofsample.CautionaryNotes:Avoidrepeatedfreezethawcycles.Anyleft-over2XEnzymeAssayReagentshouldbestoredat-20°CWITHOUTG3Ptoavoidhighbackground.

3.The5.5XDetectionAssayDiluent:(Component5):Part#3009(Magentalabel)ThawComponent5.Thisisa5.5xconcentrate.Dilutethecontents1:5.5withreagentgradeDiwatertomakeita1Xdiluent.Forexample,for100reactions,make5.5mlof1Xdiluentbyadding1mlofthe5.5XDetectionassaydiluent to4.5mLof reagentgradeDiwater.Anyunusedmaterial canbealiquotedandstoredfrozenbetween-200Cand-40°Cinpolypropylenetubes.Note: Prepare as much of the 1X Detection Reagent Diluent (Part # 3009) as needed for the day’sexperiments.Preparealiquotsofrequiredvolumeperplateandstoreat4°C.Bringtoroomtemperatureabout 10 minutes prior to addition. Add the 50X Detection Reagent (Part # 6002) to the diluentIMMEDIATELYbeforethereaction.



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Preparationof1XDetectionReagent:For250reactionson4plates,2.75mlof5.5XDetectionReagentDiluent(Part#3009:Component5)+12.375mlofdiwatermakes1XDetectionReagentDiluent Aliquot3.5mlpertubeandstoreat4°Coronice. Equilibrateeachtubefor10minutesatRT Add70µLof50XDetectionReagent(Component4:Part#6002)justbeforeaddingtoreactionwells.4.The50XDetectionReagent:(Component4):Part#6002(Magentalabel)Thisisafrozen50XconcentratecontainingLuciferaseandLuciferin.Priortouse,thawthevialandkeeponice.Aliquotassingle-useportionsinpolypropylenevialsandfreezetherestbetween-200Cand-40°C,ifyouarenotusingthecompletecontentsofthevial.Just before adding the detection reagent to your sample, dilute it 1:50 with the 1X Detection AssayDiluentprepared in step 3 above. For 100 reactions, 100µL of the 50XDetection Reagent is added to4.9mlofthe1XDetectionAssayDiluenttomake1XDetectionReagent.Add50µLofthe1XDetectionReagenttoeachsampleinthewellcontaining100µLofcells+100µLofthe2XEnzymeReactionCocktail.CautionaryNotes:1.Keepcontentsprotectedfromdirectlightatalltimes.2.Avoidrepeatedfreezethawcycles.3.Luminometersensitivity:IftheRLUvaluesarelow,itmayindicatethattheluminometerisnotverysensitive.Insuchacase,we recommend titrating theDetection reagent (Part #6002) toa1:25, 1:10anda1:5dilution in Step4above todetermineasuitablestrength.Pleasenotethatthiswill reducethenumberof reactionsthatcanbeperformedusingoneaCella-TOXkit to250,100or50testsdependingonthedilutionused.Wehavefoundthatdifferentluminometershavedifferentsensitivities.WehavetestedtheBDMonolite,Berthold,PEVictor,PETopCountandtheVeritas.TheassayhasbeenoptimizedtoworkontheVeritasluminometerfromTurnerBiosystems.5.LysisBuffer:Component6:(Part#3035)(Yellowlabel).Add10µLoflysisbufferasapositivecontrolfortotalreleaseofGAPDH.Storelysisbufferat4-80C.Equilibratetoroomtemperaturebeforeuse.Note:Forsomecelllines,itmaybenecessarytoperformatitrationofthelysisbuffertooptimizecelllysis.Usetheminimum amount of lysis buffer that gives themaximum signal in a titrationwith 10,000 cells.(SubstitutingwithTritonX100asthelyticagentwillseverelyreducetheassaysignal.)



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V.AssaysGeneralGuidelines:Cell Technology’s non-radioactive aCella-TOX kit can be used to efficiently and accurately measureAntibodyMediatedCellularCytotoxicity (ADCC),CellularMediatedCytotoxicity (CMC)andComplementdependent cytotoxicity (CDC). The following guidelines will help to ensure the success of assays usingaCella-TOX. Since the aCella-TOX kit measures the release of the GAPDH enzyme as an indicator ofADCC/CMC,itishighlyrecommendedtousefresheffectorcellswhenusingwholeblood.Peripheralbloodlymphocytes(PBL’s)containaheterogeneouspopulationofcells,manyofwhicharenot involvedintheADCC/CMCreactions.We highly recommend using freshly isolated PBL’s or fresh immunoaffinity purified effector cells (forexampleNKorCD8+T lymphocytes). Cytokine-activatedPBL’s (9,10)mayalsobeemployed in this assay.Theuseofcytokines,however,maycausetheassaytoexhibitahigherdonor-to-donorvariation(CMC)aseachdonor’sNKcellsmaybestimulated toadifferentextentby thecytokine.Theuseof10% low IgGserumhelpsreducebackgroundandincreasesensitivityoftheADCCassay.Avoidusingserumwithhighhemoglobin contamination (Hb levels <6.5mg%desirable) as thiswill increase background and reduceassaysensitivity.Avoidusingwellsontheedgeoftheplate,asthiswillincreaseassaysignalvarianceduetothe“edgeeffect”.TheseedgewellsshouldbefilledwithmediaorPBS.BeforestartingADCC/CMCexperiments,werecommendperformingthefollowingpreliminaryassays:

1. Celltitrationontargetcellstodeterminetheoptimalnumberofcellstobeplated.

2. Determinationoftheoptimaleffector:target(E:T)ratiobyusingvaryingratiosofeffectorcells(forexample,5to25)keepingthenumberoftargetcellsandantibodyconcentrationconstant.(forADCCAssays)

3. For ADCC Assays: Antibody titration to determine the dynamic range required to obtain an

optimaldose-responseintheADCCassay.

4. WerecommendadaptingthetargetcellsinlowIgGmediaforafewpassagesbeforetheassay.

5.1Targetcelltitration:

GAPDHexpressionwillvarybetweentargetcelllines.ItisimportanttodeterminethelinearresponserangeofaCella-TOXwithinyourparticulartargetcell line.Thiscanbeaccomplishedbytitratingthetargetcellsintheassaymedia(werecommend20,000to1000cells/well),addinglysisbuffertoeachwell (asdescribed inmaximum lysis controlbelow).Nextaddenzymeassay reagentanddetectionreagent as described below and measure luminescence. For further experiments, use the cellconcentrationthatfallsinthelinearrangeoftheassay.



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Seeexamplebelow:

Cell Titration

0

10000

20000

30000

40000

50000

60000

70000

0 5000 10000 15000 20000

Cell/well

RLU

Figure: Cell titrated in media, lysed and run as described above. For further assays, use cell concentration at or below 10,000cells/well.20,000cells/wellisoutoflinearrangeinthisparticularcellline.Linearity: Prolonged incubation with aCella-TOX can cause your assay to fall out of linear range. Theenzyme reagents are constantly producing ATP until one of the components becomes limiting. Takeseveraltimepointreadings5minto25mintogetanoptimizedtimepointlinearreadout.5.2DeterminationofoptimalE:Tratio:

Effectorcellsshouldbetitratedfrom5:1to25:1orgreater(dependingonthepurity).IfpurifiedNKcellsareused,E:Tof10:1or15:1couldworkwell.IfPBMC’sareused,theE:Tratiowouldhavetobehigher,possibly25or30:1.Thisshouldbedeterminedempirically,andwouldvarydependingonthetargetcellsused.Asanexample,anantibodyconcentrationof1μg/mlisappropriateforthisexperiment.Appropriate controlswouldbemedia alone, target cells alone and control including themaximumlysis of the target cells. For each effector: target (E:T) ratio, controls with effectors alone in theappropriatenumberandazeroantibodycontrol(forCMC)havetobeincludedaswell.Asampletemplatewouldbeasfollows:

1 2 3 4 5 6 7 8 9 10 11 12A B 2E ADCC

2ECMC2E

5E ADCC5E

CMC5E

10E ADCC10E

CMC10E

C 2E ADCC2E

CMC2E

5E ADCC5E

CMC5E

10E ADCC10E

CMC10E

D 2E ADCC2E

CMC2E

5E ADCC5E

CMC5E

10E ADCC10E

CMC10E

E 15E ADCC15E

CMC15E

20E ADCC20E

CMC20E

targets Maxlysis

media

F 15E ADCC15E

CMC15E

20E ADCC20E

CMC20E

targets Maxlysis

media

G 15E ADCC CMC 20E ADCC CMC targets Max media



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15E 15E 20E 20E lysisH

5.3Antibodytitration:Prepare1:4or1:5 serialdilutionsof theantibody tobe tested tocoveradynamic rangeover fourordersofmagnitude.Asanexample,astartingpointcouldbe10μg/ml.The E:T ratio as determined from the experiment above should be used. All appropriate controlsshouldbeincludedintriplicates.(targetsalone,effectorsalone,mediaandmaximumlysisoftargets).



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VI.AntibodyDependentCellularCytotoxicity(ADCC)PROCEDURE6.1SchematicRepresentationofanADCCAssay:

+37ºC15’37ºC2-4hrsTargettoEffectorcellratios:As outlined in section V above, we recommend using 5,000-10,000 target cells per well with aeffector:target cell ratio between 10:1 to 20:1 (E:T ratio as determinedby the experiments detailed inSectionVabove).SincetheaCella-TOXassay isverysensitive, it is recommendedtouse lowerratiosofeffector:targetcells;however,eachinvestigatorshoulddeterminethisempirically.6.2FortargetcellsinSuspension:A.ADCCprotocoloverview:

1. Platethetargetcellsat5000cellsperwellin25µL.2. Add25µLofantibodytothetargetcellsandincubateina37°Cincubatorfor15minutes.3. Add25µLofeffectorcellsandincubatetheplatefor2-4hoursina37°Cincubator.4. ProceedwiththeaCella-TOXreaction.

B.Preparationoftargetcells:

1. On the morning of the experiment, spin down 8-10 ml of target cells. Wash once in PBScontainingEDTA.Re-suspendinADCCculturemediacontaining10%low-IgGserum.

2. Check cell count with Trypan Blue stain and adjust cells to a concentration of 2X105 cells/ml.

Plating25µLofthiswillgive5,000cells/well.Cellconcentrationcanbeadjustedtoplatecellsatalowerorhigherconcentration.

Targetcells

Antibody

AddEffectorcells(PBMC’sorNKcells)

DetermineADCCusingaCella-TOX



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C.SeparationofPBMC’sfromleukopackorLRSchamber: IfusinganLRSchamber,thebloodshouldbedilutedwith2XvolumeofPBS/BSA/EDTAbeforeloading

ontoanequalvolumeofthedensitygradient.

1. Pipette20-25mlofHistopaque(orFicollPaqueorothersuitabledensitygradientmedium)intoa50mltube.Layer20-25mlofdiluted leukopackontheHistopaque.Centrifugeat500xgfor30minutes.(slowstop;nobrakes)

2. Carefullypipettethetopserumlayerintoaseparatetube.Aspiratethemiddlelayercontaining

the PBMCs into another tube.Wash the PBMC’swith 1-2 volumes of 1X PBS containing EDTAtwice.Centrifugeat500xgfor15minutes.

3. Repeat step2.After the finalwash, decant the supernatant and gently vortex the cells pellet.

ProceedtostepD(LysisofRedBloodCells)onlyifusingPBMC’sfortheassay.IfusingNKcells,youmayskipthisstep.

D.LysisofRedBloodCells.(pleaseskipthisstepifisolatingNKcells)

1. Re-suspendcellpellet in10-20mlof0.2%NaCl for30 seconds to lyseRBCs (gentlyagitate thetube to ensure propermixing) and immediately add 10-20ml (equal volume) of 1.6%NaCl toneutralizetheosmolarityofthecells.Donotexceed30secondsin0.2%NaCl.Aftertheadditionofthe1.6%NaCl,proceedtothecentrifugationstepimmediately.

2. Centrifugeat500xg for5-10minutesand re-suspendpellet in2 to5mlofADCCculturemedia

containing10%low-IgGserum.3. CountcellswithTrypanblueata1:100dilution.AtthistimepointisolatedPBMC’scanbeused

directlyintheADCCassayorincubatedovernightintheADCCculturemedia.Note:Dependingonthedonor,itispossiblethattheRBClysisstepmayhavetoberepeatediftheRBCcount is too high. If the supernatant is cloudy, repeat the wash step with PBS/EDTA to remove theremainingplatelets.

E.NKcellisolation:

We recommend theuseofNK cells isolatedusingCD56beadsbyPositive selection inADCCassays(usingMiltenyiBioteckitCat#130-050-401).IsolatedNKcellscanbeusedimmediatelyorculturedovernight.TheuseofisolatedNKcellsgivesabetterdose-responsecurveduetoalowerbackground,andtherefore,ahighersignal-to-noiseratio.

http://www.miltenyibiotec.com/download/datasheets_en/58/DS130_050_401.pdf

Adjusteffector cell concentration todeliver, in25µL, theoptimizedE:T ratio. If culturing cellsovernightwashthecellsinADCCmediaatthetimeoftheassay.



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F.PreparationofAntibody(e.g.,Rituximab(HumanizedAnti-CD20)):

1. Dilutetheantibodyto3µg/ml inADCCculturemedia.Nextseriallydilutetheantibody5-fold7times across the plate. Plating 25µl of each antibody concentrationperwellwill yield a rangefrom 1ug/ml to 6.4X10-5µg/ml (final concentrations). Starting antibody concentrations are 3Xsincetheywillbesubsequentlydiluted1in3bytheadditionoftargetandeffectorcells.

2. Forthezero-antibodycontrol,plate25µLofmediaonly(seesectionG.Control5below).Note:Performreactionsintriplicate.

G.Controls:

1. Control 1: Spontaneous release of GAPDH from target cells: Plate 25µL containing 5,000targetcellsintriplicateandadd50µlofADCCmedia.

2. Control2: Spontaneous releaseofGAPDHbyeffector cells:Plate25µL containingeffectorcells(thesameratioasusedintheexperiment)andadd50µLofADCCmediaintriplicate.

3. Control3:Maximumlysisoftargetcells:Plate25µLcontaining5,000targetcellsintriplicateandadd50µLofADCCmedia.

4. Control4:Mediaonlycontrol:Plate75µloftheADCCmediaintriplicate.5. Control 5: Asmentioned above, a zero Antibody control, 25uL ofmedia+ effector cells +

targetcells.Thisreactionrepresentscell-mediatedcytotoxicity(CMC)thatoccursduetothekillingofthetargetsbytheeffectorswithoutthemediationoftheantibody.

H.PreparationofaCella-TOXreagents:ThereagentssuppliedintheaCella-TOXkitarelightsensitive.Avoiddirectlight,asinabio-safetyhood.Indirect lab light is acceptable for short exposures (15-20 minutes). Reduce light levels in the lab ifpossible.Follow the step-by-step directions described in the section “aCella-TOX: Reagent Set Up” (Section 4.5above)oftheprotocolsuppliedwiththekittomakeworkingstocksofreagents.ThereagentsshouldbeatequilibratedtoroomtemperaturebeforeaddingthemtotheADCCassay.I.ADCCReaction:

1. In setting up the plate, plan to avoid using the outer rows and columns to reduce any “edgeeffects”.Addonly200µLofADCCculturemediaorPBStothesewells.

2. The target and effector cells should be counted and set up at required cell counts. The serialdilutionsoftheantibodyshouldbereadyforuseintherequiredvolumes.

3. Platethetargetcellsat5000cells/well in25µLofmedia intriplicate inasterile,U-bottom,96-welltissuecultureplate.

4. Setupallcontrolsasmentionedabove.5. Plate25µLofeachdilutionoftheantibodyinthewellscontainingthetargetcells.6. Incubate the plate at 37oC for 15 minutes to allow opsonization of antibody to occur. (This

optionalstepshouldbeincludedbasedonthetargetcellline.)7. Add25µLoftheeffectorcellstostartthereaction.Spindownplatefor1minuteat500gtobring

targetsandeffectorsinclosecontact.8. Covertheplatewitha96-wellplatecover.Incubatetheplateat37oCfor2-4hours.Thetimeof

thereactionshouldbedeterminedempirically.9. Attheendoftheincubation,allowtheplatetoequilibratetoRTfor15minutes.

Add10µLoflysisbuffer(Component6,Part#3035)tothewellsindicatedformaximumlysisoftargetcells.Incubatefor5-10minutesatRT.(monitorlysisundermicroscope)



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10. Add125µLoflow-IgGserummediatoallthereactionwellstobringthevolumeto200µL.Spindownplateinamicroplatecentrifugefor1’at750Xg.

11. Inanopaqueplatefortheluminometer(PerkinElmerOptiplates#6005290),add50µLofEnzymeAssay Diluent (Component 2, Part #3008) to all corresponding reaction wells. Now carefullytransfer50µLofthesupernatantto50µLofComponent2usingamultichannelpipette,changingtipseachtime.

12. Add100µLoftheEnzymeAssayreagentcontainingG3P(fromstepIV:Proceduresabove)toallreactionwells.

13. Immediately,add50µLoftheDetectionreagent(fromstepIV:proceduresabove)toeachwell.14. Readtheplateontheluminometerimmediately(withoutthecover)andtakeseveraltimepoint

readings5minutesaparttodeterminetheoptimalreadingtime-point.J.Calculations:TodetermineADCC,firstcalculatethemeanofthetriplicateluminescentvaluesforeachofthereactionconditions.Firstsubtractthemeanmediaonly luminescentvalue(control4) fromallcalculatedmeanvalues.Thenusethefollowingformulatocalculate%ADCC.%ADCC=(Sample)-(Control1Targetspontaneousrelease)-(Control2effectoralone)X100

(Control3maximumrelease)-(Control1Targetspontaneousrelease)

Theequationabovenormalizesthedatafor%ADCCbysubtractingthespontaneoustargetreleasefromthemaximumlysiscontrol.For the dose-response curve, plot the log of the Antibody concentrations inmg/ml vs. the%ADCC ascalculatedbytheformulaabove.

Rituxan 2 hours

0.000010.0001 0.001 0.01 0.1 1 10

20

40

60

80

100E:T 10:1E:T 20:1

log Rituxan ug/ml

%AD

CC

+/-S

D

Rituxan 3.5 hrs

0.0001 0.001 0.01 0.1 1 1020

40

60

80

100E:T 10:1

log Rituxan ug/mL

%AD

CC

+/-S

D

Figure:5000Ramoscells/wellwereincubatedwithseriallydilutedRituxanantibodyfor15minutespriortotheadditionofpurifiedNK cells stimulatedovernightwith 10ng/ml IL-2. TheADCC reactionwas further incubated for 2 hours (top graph) or 3.5 hours(bottomgraph)at thespecifiedE:T ratios.%Cytotoxicitywasmeasuredusing theaCella-TOXassay.3.5hour timepointhasbeenoptimizedwiththe10:1E:Tratio.Graphwith20:1E:Tratioisflat.(100%cytotoxicity)



16

Daudi T=18, 2 hrs

Rituxan ug/mL

%AD

CC

0.000010.0001 0.001 0.01 0.1 1 100

20

40

60

80% ADCC 25 to1 E:T%ADCC 10 to 1 E:T

Figure:5000Daudicells/wellwereincubatedwithseriallydilutedRituximabfor15minutespriortotheadditionofpurifiedNKcellsstimulated overnight with IL-2. The ADCC reaction was further incubated for 2 hours at the specified E:T ratios. % ADCC wasmeasuredusingtheaCella-TOXassay.Log(EC50)valueforE:T25:1was-2.344andforE:T10:1was-2.213.K.TechnicalNotes:

1. Thevolumeoflyticagentper75µLofcellstoobtain100%lysishastobedeterminedempirically,bycheckingunderamicroscope.Werecommendstartingat10mLandtitratingdown(lysisbuffercanbe titrated indiwater). The incubation timecouldalsovary from5-15minutes. Theplateshould be allowed to equilibrate to RT for about 15 minutes before the addition of the lysisbuffertothemaximumlysiswells.

2. ItisimportanttousefreshlyseparatedPBMC’sfrombloodinordertoreducedeathofeffectorcells,whichwill increase effector-only background.Also, highRBC contamination in thePBMCfractionwillincreasebackgroundandcompromiseassaysensitivity.

3. ForNKcellisolation,werecommendavoidingtheRBClysisstep.ThismayhelpimprovethefinalyieldoftheNKcellsisolatedperdonor.

4. TheoptimaltargettoeffectorcellratioalsohastobedeterminedpriortoperformingtheADCCassay.Platingtwoorthreedifferenttargetcellconcentrationsandvaryingtheeffectorcellratiosacrosstheplatecanaccomplishthis.

5. Thereisconsiderablevariationintheeffectorcells isolatedfromdifferentdonors.Thiseffect isamplifiedifusingcytokinestomaintainthePBMC’sorNKcells.WerecommendusingtheADCCculturemediaifeffectorcellshavetobemaintainedovernight.

6. We strongly recommend theuseofNK cells inADCC assays (isolatedusingMiltenyi Biotec kitCat# 130-050-401). Isolated NK cells can be used immediately or cultured overnight in a CO2

incubator. The use of isolated NK cells gives a better dose-response curve due to a lowerbackground,andtherefore,ahighersignal-to-noiseratio.

7. Targetcellsshouldnotbeextensivelysubcultured(followguidelinesspecifictocellline),astheymayexhibitchanges in levelsofsurfaceantigenexpression,whichmaymakethemresistanttocytotoxicitybytheantibody.

Note:When choosinganoptimized timepoint for your calculations, it is important to choosea timepointwherethemaximumlysiscontrolisincreasinginRLU’s.(Typically,weuseT=15’).Donotchooseatimepointwherethemaximumlysishasdecreasedfromtheprevioustimepoint.AschematicrepresentationofanADCCreactionplateisshownbelow:Usingtwopreviouslydeterminedeffector:targetcellratios,setupanantibodytitrationacrosstheplatewiththeappropriatecontrols.Reactionsarerunintriplicates.



17

1 2 3 4 5 6 7 8 9 10 11 12

A

B Antibody1ug/ml

Antibody0.2ug/ml

Antibody0.04ug/ml

Antibody0.008ug/ml

Antibody0.0016ug/ml

Antibody0.00032ug/ml

Antibody0ug/mlControl5

E:T10:1Control

2

Control1

C Antibody1ug/ml

Antibody0.2ug/ml

Antibody0.04ug/ml

Antibody0.008ug/ml

Antibody0.0016ug/ml



E:T10:1Control

2

Control1

D Antibody1ug/ml

Antibody0.2ug/ml

Antibody0.04ug/ml

Antibody0.008ug/ml

Antibody0.0016ug/ml



E:T10:1Control

2

Control1

E Antibody1ug/ml

Antibody0.2ug/ml

Antibody0.04ug/ml

Antibody0.008ug/ml

Antibody0.0016ug/ml



E:T20:1Control

2

Control3

Control4

F Antibody1ug/ml

Antibody0.2ug/ml

Antibody0.04ug/ml

Antibody0.008ug/ml

Antibody0.0016ug/ml



E:T20:1Control

2

Control3

Control4

G Antibody1ug/ml

Antibody0.2ug/ml

Antibody0.04ug/ml

Antibody0.008ug/ml

Antibody0.0016ug/ml



E:T20:1Control

2

Control3

Control4

H

6.3ADCCwithAdherentTargetCellLinesA. ProtocolOverview:

1. Platethetargetcellsat5000cells/wellthenightbeforetheassay.2. Washwith freshmedia and decant. Add 50μL of the antibody directly on the cells and allow

opsonizationfor15minutes.3. NKcellspurifiedfromaleukopack.Theyarewashedthemorningoftheassayandaddedtothe

targetcellsinavolumeof50μL.Theplateisincubatedfor2-4hours.4. PerformtheaCella-TOXreaction.

B. Preparationoftargetcells:1. The night before the assay, the cells are trypsinized, washed and plated at the required cell

density (5000 cells/well) in 100μL of low IgG-serum containingmedia on a U bottom 96-wellTissuecultureplate.NOTE:Allreactionsshouldbedoneintriplicates.For certain cell lines, itmay be required to use a gentler Trypsin replacement such as TrypLEExpress(catalog#12604fromGIBCO).

2. Incubateovernightat37ºC.3. Onthemorningoftheassay,washthecellswithabout100μLoffreshwarmmediaoncebefore

the addition of the antibody. This can be accomplishedmy gently inverting the tissue cultureplateoverapapertowelandgentlytappingittodecantthemedia.

4. Nextgentlyadd100μLoffreshmediaanddecantthemediaasdescribedabove.



18

C. Preparationofantibodydilutions:

1. Startingwitha concentrationof about1μg/ml (determined from titrationperformed inearlierexperiment),prepareserial2XdilutionsoftheAntibody(asdesired)torangeoverthreeordersofmagnitude.Thesedilutionsarepreparedinthelow-IgGserummedia.

2. Decant themedia from theplateandadd theantibodydilution in50μl volumedirectly to thewashedcells.

3. Incubateat37ºCfor15minutesforopsonizationtooccur.4. For the zeroAntibody control (CMC) reaction, 50μLofmedia is added to the target cells (see

sectionE.Control5below)

D. PreparationofEffectorcells:Refer toSectionC:SeparationofPBMC’s fromLeukopackandSectionD:LysisofRedBloodCellsabove.PBMC’smaybeusedimmediatelyormaintainedintheADCCculturemediaovernightat37°C.PBMC’s can be further sorted and specific populations; such as NK cells may be isolated usingMiltenyiBiotec’sMACSsystems.Wefindthisreducestheeffectorcellbackgroundsignificantly.http://www.miltenyibiotec.com/download/datasheets_en/58/DS130_050_401.pdf1. Onthemorningoftheassay,theeffectorsmustbespundown,washedwithfreshwarmmedia,

and counted. Effectors should be resuspended in ADCC media at the appropriate cellconcentrationtodelivertheoptimalE:Tratioin50μL.

2. Incubatetheplatefor2-4hoursat37ºCfortheADCCreactiontooccur.E. Controls:

1. Control1:SpontaneousreleaseofGAPDHfromtargetcells:Towellscontaining5,000targetcellsintriplicate,add100µlofADCCmedia.

2. Control2:SpontaneousreleaseofGAPDHbyeffectorcells:Plate50µLofeffectorcells(thesameratioasusedintheexperiment)andadd50µLofADCCmediaintriplicate.

3. Control3:Maximumlysisoftargetcells:To5,000targetcells intriplicate,add100µLofADCCmedia.

4. Control4:Mediaonlycontrol:Plate100µloftheADCCmediaintriplicate.5. Control5:Asmentionedabove,azeroAntibodycontrol,50μLofmedia+50μLofeffectorcells

shouldbeaddedtotargetcells,whicharealreadypresent inthewell.Thisreactionrepresentscell-mediated cytotoxicity (CMC) that occurs due to the killing of the targets by the effectorswithoutthemediationoftheantibody.

F. PreparationofaCella-TOXreagents:ThereagentssuppliedintheaCella-TOXkitarelightsensitive.Avoiddirectlight,asinabio-safetyhood.Indirect lab light is acceptable for short exposures (15-20 minutes). Reduce light levels in the lab ifpossible.Followthestep-by-stepdirectionsdescribedinthesection“aCella-TOX:ReagentSetUp”(above)oftheprotocolsuppliedwiththekittomakeworkingstocksofreagents.ThereagentsshouldbeatequilibratedtoroomtemperaturebeforeaddingthemtotheADCCassay.



19

G. ADCCReaction:

1. In setting up the plate, plan to avoid using the outer rows and columns to reduce any “edgeeffects”.Add200µLofADCCculturemediaorPBStothesewells.

2. The target and effector cells should be counted and set up at required cell counts. The serialdilutionsoftheantibodyshouldbereadyforuseintherequiredvolumes.

3. Washedtargetcellsareincubatedwiththeantibody(50μL)for15minutes,allowingopsonizationtooccur.

4. Effectorcells(NKcellsorPBMC’s)areaddedinavolumeof50μL.TheADCCreactionisallowedtoproceedfor2-4hoursat37ºC.

5. Attheendof the incubation, thereactionplate isallowedtoequilibrate toRT for15minutes.LysethetargetcellsintheMaximumLysiscontrolwellswith10μLofLysisbuffer.Incubate5-10minutesatRT.(monitorlysisundermicroscope)

6. Add100μLoflowIgG-serummediatoallreactionwellstobringvolumeto200μL.7. Spindownplateinamicroplatecentrifugefor1’at750Xg.8. In the opaque plate for the luminometer, (Perkin Elmer Optiplates#6005290) add 50μl of

Component2(EnzymeAssayDiluent,Part#3008)toallreactionwells.9. Nowcarefullytransfer50μLofthesupernatanttothe50μLofComponent2usingamultichannel

pipettor,changingtipseachtime.10. Add100μLof the2XEnzymeAssay reagentcontainingG3P (fromstep IV:Procedures) toeach

reaction.11. Immediately,add50μLof1XDetectionreagent(fromstepIV:Proceduresabove)toeachwell.12. Readtheplateontheluminometerimmediately(withoutthecover)andtakeseveraltimepoint

readings5minutesaparttodeterminetheoptimalreadingtime-point.13. Analyzeandgraphdata.

H.Calculations:

TodetermineADCC,firstcalculatethemeanofthetriplicateluminescentvaluesforeachofthereactionconditions.Firstsubtractthemeanmediaonlyluminescentvalue(control4)fromallcalculatedmeanvalues.Thenusethefollowingformulatocalculate%ADCC.

%ADCC=(Sample)-(Control1Targetspontaneousrelease)-(Control2effectoralone)X100

(Control3maximumrelease)-(Control1targetspontaneousrelease)Theequationabovenormalizesthedatafor%ADCCbysubtractingthespontaneoustargetreleasefromthemaximumlysiscontrol.

For the dose-response curve, plot the log of the Antibody concentrations inmg/ml vs. the%ADCCascalculatedbytheformulaabove.

Note:When choosinganoptimized timepoint for your calculations, it is important to choosea timepointwhere themaximum lysis control is increasing inRLU’s.Donot choosea timepointwhere themaximumlysishasdecreasedfromtheprevioustimepoint.(Typically,weuseT=15’)



20

AschematicrepresentationofanADCCreactionplatefortwoantibodies(positiveandnegative)isshownbelow:(1:4serialdilutionsofantibodies) 1 2 3 4 5 6 7 8 9 10 11 12A B 20μg/ml 5μg/ml 1.25 0.3125 0.078 0.0195 0.0049 0 T E C +antibody 0 T E D triplicates 0 T E E 20μg/ml 5μg/ml 1.25 0.3125 0.078 0.0195 0.0049 0 Max Media F -antibody 0 Max Media G triplicates 0 Max Media



21

VII.CellMediatedCytotoxicity(CMC)Procedure7.1SchematicRepresentationofaCMCAssay +37ºC2-4hrs7.2CMCAssayDescriptionA.Cells,Reagentsandinstrumentsrequired:

1. TargetcellssuchasK562,Jurkatsorothercellline.2. Leukopack,LRSchamberorBuffypack(sourceoffreshblood)3. CellCulturemedia(RPMI1640supplementedwith10%FBS,Non-EssentialAminoacidsand

Penn/Strep+L-Glutamine).4. Histopaque,Sigma#10775. aCella-TOXKit(CellTechnology,Inc.)6. Luminometer7. Whiteopaque96-wellOptiplates(PerkinElmer,#6005290orCorning#3605withcover,#3099)8. Ubottom96welltissuecultureplatesforCMCreation.9. Sterile-filteredsolutionsof0.2%NaCland1.6%NaClforRBClysis.10. Sterile-filteredsolutionof!XPBScontaining2mMEDTA.11. MiltenyiBiotecMidiMACSStartingkit,(Cat.#130-042-301)includingMidiMACSseparationunit,

1MACSMultistand,25LScolumnsandCD56Microbeads(Cat.#130-050-401).B.Preparationoftargetcells:

1. Onthemorningoftheexperiment,centrifuge8-10mlofTargetcells.Washoncein1XPBScontainingEDTA.Resuspendinculturemedia.

2. CheckcellcountwithTrypanBluestainandadjustcellstoaconcentrationof2X105cells/ml.

Plating50µLofthiswillgive10,000cells/well.Cellconcentrationcanbeadjustedtoplatealowercellnumber.

Targetcells

AddEffectorcells(PBMC’sorNKcells)

DetermineCMCusingaCella-TOX



22

C.SeparationofPBMC’sfromleukopack:If using an LRS chamber, theblood shouldbedilutedwith2X volumeofPBS/BSA/EDTAbefore loadingontoanequalvolumeofthedensitygradient.

1. Pipette 20-25mlofHistopaque (or other suitabledensity gradientmedium) into a 50ml tube.Layer20mlofbloodontheHistopaque.Centrifugeat500xgfor30minutes.

2. Carefully pipette out the top serum layer into a separate tube. Aspirate the middle layer

containing the PBMCs into another tube, taking care tominimizeRBC contamination. Add 1-2volumes of PBS containing EDTA to the PBMC’s and centrifuge at 500xg for 15minutes. NextdecantthePBMC’sandgentlyvortexthecellpellet.

3. Repeatwashstep2.Afterthefinalwashdecantsupernatantandgentlyvortexthecellpellet.

D.LysisofRedBloodCells:

1. Resuspendcellpelletin10-20mlof0.2%NaClfor30secondstolysetheRBCs(gentlyagitatethetube to ensure propermixing) and immediately add 10-20ml (equal volume) of 1.6%NaCl toneutralizetheosmolarity.Donotexceed30secondsinthe0.2%NaClsolution.Aftertheadditionofthe1.6%NaCl,proceedtothecentrifugationstepimmediately.

2. Centrifuge for 10 minutes and resuspend the cell pellet in 2 to 5ml of ADCC culture media

containing10%lowIgGserum.Note:Dependingonthedonor, it ispossiblethattheRBClysisstepmayhavetoberepeatediftheRBCcount is too high. If the supernatant is cloudy, repeat the wash step with PBS/EDTA to remove theremainingplateletsE.PreparationofEffectorcells:

PBMC’smaybeusedimmediatelyoractivatedwithIL-2overnight.PBMC’scanbefurthersortedandspecificpopulations;suchasNKcellsmaybeisolatedusingMiltenyiBiotec’sMACSsystems.Wefindthis reduces the effector cell background significantly. We recommend isolating NK cells usingMiltenyiBiotec’skitcat#130-050-401.http://www.miltenyibiotec.com/download/datasheets_en/58/DS130_050_401.pdf

1. On themorning of the assay, the effectorsmust be spundown,washedwith freshwarm

media,andcountedwithTrypanBlue.Effectorsshouldbe resuspended in theappropriatevolumeofmediatodelivertheoptimalratiotothetargetcellnumberin50µL.SinceaCella-TOXisaverysensitiveassay,startwitha1:1ratioandtitrateup.

2. Immediately, spin down the plate for 1 minute at 500Xg to allow surface contact of the

EffectorsandTargets.(onlyforsuspensioncells.Donotspiniftargetcellsareadherent)

3. Incubatetheplatefor2-4hoursat37ºC.F.Controls:

1. Control1:Targetcellsalone:spontaneousreleaseofGAPDH.Plate50µLoftargetcells+50µLofmedia.

2. Control2:EffectorcellsaloneattherespectiveE:Tratios.:spontaneousreleaseofGAPDHfromeffectors.Plate50µLofeffectorcells+50µLofmedia.



23

3. Control3:TargetcellsaloneforMaximumGAPDHrelease.Plate50µLoftargetcells+50µLofmedia.

4. Control4:Mediaonly.Plate100µLofmedia.G.PreparationofaCella-TOXreagents:ThereagentssuppliedintheaCella-TOXkitarelightsensitive.Avoiddirectlight,asinabio-safetyhood.Indirect lab light is acceptable for short exposures (15-20 minutes). Reduce light levels in the lab ifpossible.Followthestep-by-stepdirectionsdescribedinthesection“aCella-TOX:ReagentSetUp”(above)oftheprotocolsuppliedwiththekittomakeworkingstocksofreagents.ThereagentsshouldbeequilibratedtoroomtemperaturebeforeaddingthemtotheADCCassayH.CMCReaction:Plateallreactionsintriplicate.

1. Plate5000targetcellsin50µL+50µLofthevariouseffectorcellnumbers.Platethevariouscontrolsasshowninthetemplate.

1. In the case of suspension target cells, the platemay be spun down for aminute to establishcontactbetweenthetargetsandeffectors.Wedonotrecommendadherentcellsbespundown.

2. CovertheplateandincubatetheCMCreactionat37oCfor2-4hours.Thislengthoftimeshouldbedeterminedempirically.

3. Allow the plate to equilibrate to RT for 15 minutes. Add 5-10µl of lytic agent to the wellsindicatedformaximumlysisoftargetcells(control3).Incubate5-10minutesatRT.(monitorlysisundermicroscope)

4. Add100µLofmedia toall reactionwells tobringvolumeto200µL.Spindownforaminuteat750Xg.

5. Transfer50µLsupernatantintocorrespondingwellsonawhiteplatecontaining50µLofEnzymeassaydiluent.

6. Add100µLoftheEnzymeAssayreagentcontainingG3Ptoallreactionwells.7. Immediately,add50µLoftheDetectionreagenttoeachwell.8. Readtheplateontheluminometer(withoutthecover)immediatelyandtakeseveraltimepoint

readings5minutesapart,todetermineoptimalreadingtime-point.I.Calculations:In order to calculate % cytotoxicity, calculate the mean of the triplicate luminescent values for eachreactionconditionandcontrolsandusethefollowingprocedures:

a. First subtract the meanmedia only luminescent value (control 4) from all calculatedmeanvaluesforeachreactioncondition.

b. Next for each calculated mean value of E:T ratio, subtract the respective meancalculatedvalueofeffectorspontaneousreleasecontrol2.

c. Thisisrepresentedbythefollowingformula:%cytotoxicity=(Sample)-(Control1Targetspontaneousrelease)-(Control2Effectorsalone)X100 (Control3maximumrelease)-(Control1Targetspontaneousrelease)Forthedose-responsecurveplottheE:Tratiovs.the%cytotoxicityascalculatedbytheformulaabove.



24

Cell Mediated Cytotoxicity K562 cells

0.1 1 10 1000

20

40

60

80

Effector to Target Ratio

% C

ytot

oxic

ity

FigureshowsCMCassaywithK562astargetcellsincubatedwithdifferentE:TratiosofIL-2stimulated(overnight)NKcellsisolatedusingMiltenyiBiotec’skitcat#130-050-401.Targetcellswereplatedat5,000cells/wellandincreasingeffectorcellconcentrationsfrom1:1toaratioof32:1.Theassaywasincubatedfor2hoursat37°Cand%cytotoxicitymeasuredwiththeaCella-TOXkit.

Cell Mediated Cytotoxicity

0.00

2.00

4.00

6.00

8.00

10.00

12.00

0 5 10 15 20 25 30 35

E:T Ratio

% C

ytot

oxic

ity

Figure:CMC:10,000Jurkatcells/wellweremixedwithfreshlyisolatedPeripheralBloodLymphocytesatvariousEffector:Targetcellratios.TheCMCassaywasincubatedfor4hoursand%cytotoxicitydetectedusingtheaCella-TOXassay.



25

SampleCMCsetup 1 2 3 4 5 6 7 8 9 10 11 12

A

B E:T1:1

E:T1:1

E:T1:1

Control21E

Control21E

Control21E

Control1

Control4

C E:T2:1

E:T2:1

E:T2:1

Control22E

Control22E

Control22E

Control1

Control4

D E:T4:1

E:T4:1

E:T4:1

Control24E

Control24E

Control24E

Control1

Control4

E E:T8:1

E:T8:1

E:T8:1

Control28E

Control28E

Control28E

Control3

F E:T16:1

E:T16:1

E:T16:1

Control2

16E

Control2

16E

Control2

16E

Control3

G E:T32:1

E:T32:1

E:T32:1

Control2

32E

Control2

32E

Control2

32E

Control3

H

TECHNICALNOTES:1.HighRBCcontaminationinthePBMCfraction(effectorcells)willincreasebackgroundandcompromiseassaysensitivity.2. Substituting regular FBS with low IgG serum (Gibco, Cat. # 16250)may help to lower the standarddeviations.3.Differentdonors reactdifferently to IL-2andother cytokines. Theuseof cytokineswith theeffectorcellsmaythereforecontributetoahigherCMCresponse,whileaddingtothedonor-to-donorvariability.4.We recommendusing purifiedNK cells instead of PBMC’s as effector cells to increase the signal-to-noiseratio. IfNKcellsareused in theassay,a lowerE:T ratio (typically10:1) than isused forPBMCs isrecommended.



26

VIII.ComplementDependentCytotoxicity(CDC)

+37ºC15minutes37ºC30minutes-2hours

AntibodyDependentComplementMediatedCytotoxicityProtocol:MaterialsRequired:1.96willtissuecultureplates,Whiteopaque96wellplates.2.Mediaofchoice.3.Labinstruments,pipettes,tipsetc.Assay:1.Platetargetcellsbetween5,000-10,000,in25-50μL,cellsperwellincompletemedia(heat-inactivated10%FCS+Penn/Strep/L-Glutamine).Use96welltissuecultureplates.2. Titrate serial dilutionsof antibody, in25μL,onto the target cells (include zeroantibody control) andincubate15minutesat37°CinCO2incubatorforopsonizationtooccur.3.Addcomplementin25μL,toafinalconcentrationof5-25%(thisneedstobedeterminedempiricallyasperyourcomplementsource.)4.Controls:

A.Maximumlysis(Targetcells+complement+media).5-10μLoflysisbuffer(Part#3035)willbeaddedtothiscontrol.B.Targetspontaneousrelease(nocomplement).Nolyticagentwillbeaddedtothiscontrol.C.Mediacontrolwithcomplement.

5.Setupsamplesandcontrolsandincubatethereactionplatefor30-120minutesinincubatorat37°C.This incubation time will depend on antibody and complement source and must be determinedempirically.

Targetcells

Antibody

AddComplement

Determine%cytotoxicityusing

aCella-TOX



27

6.Afterrequiredincubationtime,removethe96wellplatefromtheincubatorandallowittoequilibratetoroomtemperature(15minutes).7.Add5-10μLoflysisbuffer(Part#3035)tothemaximumlysiscontrolwells(controlA).Pipetteupanddownseveraltimestoensurecompletelysis.Incubatefor5minutesatRT.8.Nextadd100μLor125μLofmediatoallwellstobringthefinalvolumeto200μL.9.Spindowntheassayplate(1minuteat750Xg).10.Transfer50μLsupernatantintocorrespondingwellsonawhiteplatecontaining50μLofEnzymeassaydiluent.(Part#3008)11.Add100µLofthe2XEnzymeAssayreagent(Part#6001+Part#3008)containingG3P(Part#6003)toallreactionwells.

12. Immediately,add50µLofthe1XDetectionreagent(Part#6002+dilutedPart#3009)toeachwell.Note:Followthestep-by-stepdirectionsdescribedinthesection“aCella-TOX:ReagentSetUp”(Section4.5above)tomakeworkingstocksofreagents.

13. Readtheplateontheluminometer(withoutthecover)immediatelyandtakeseveraltimepointreadings5minutesapart,todetermineoptimalreadingtime-point.(typicallyT=15)

Calculations:Inordertocalculate%cytotoxicity,calculatethemeanofthetriplicateluminescentvaluesforeachreactionconditionandcontrolsandusethefollowingprocedures:

a. AveragecontrolsA,BandC.b. Firstsubtractthemeanmediaonlyluminescentvalue(controlC)fromallsamplewells

andfromcontrolsAandB.c. Thisisrepresentedbythefollowingformula:

%cytotoxicity=(SampleRLU’s)-(ControlBTargetspontaneousrelease)X100 (ControlAmaximumrelease)Forthedose-responsecurveplottheE:Tratiovs.the%cytotoxicityascalculatedbytheformulaabove.



28

Figure:TheabovegraphshowsanantibodytitrationofRituximabfrom0.000977to1μg/mlincubatedwith5%babyrabbitcomplementand5000Dauditargetcellsfor30minutesat37oC.

IX.DrugInducedCytotoxicityA.Recommendedcontrols:

1.NegativeControl:In100µL:A. Plateintriplicatecellculturemediaalonetomeasurebackgroundsignal.

AND/ORB. Untreatedcellsorcellstreatedwithsolvent/vehicletodissolvetestcompound.

2.PositiveControl:MaximumReleaseofGAPDH:A.Plateintriplicatecellsalone.Attheendoftheexperimentalprotocoladd5-10µLofthelysisbufferto100µLofmaximumreleaseofGAPDHcontrol.Note: Use the optimized volume of lysis buffer for your cell line as determined in StepIV:Proceduresstep5reagentsetup(above).

B.Assay:Note:

1. Washing the cells into fresh media prior to starting the assay could result in lowering thebackgroundsignal.

2. Wehavefoundthatthebackgroundsignalinthisassaygreatlydependsonthetypeofserumusedinthecultureofcells.WerecommendtheuseofUSDAapproved,USorigin,FetalBovineserawithHemoglobinlevels<6.5mg%,inordertogetlowestbackgroundsignals.



29

1. Inopaquewhiteplates,plateintriplicate,testsamplecellsat1000-10,000cellsperwell in100µL.Thenplateallcontrolsin100µL.Cellsmaybeplatedinreducedserumor10%serummedia.Wehavedetermined the linear rangeof theassay in thepresenceof10%FCS tobebetween1000 and 20000 cells/well. In serum-freemedia, aCella-TOX can detect as few as 7 cells/well.Therefore,thepresenceofserumcompromisestheassaysensitivity.Itisimportanttodeterminetherangeofcellconcentrationinordertomaintainthelinearresponseforaparticularcellline.

2. Addtestcompoundstoyoursamplecells.

3. Afterincubationaccordingtoyourexperimentalprotocol:

A. Maximum Lysis (control 3): To 100µL of the positive control sample, add 5-10µL of lysisbuffer.Incubate5-15minutesbeforeproceedingtostepB.

B. Reaction: Then add 100µL of 2X Enzyme Reaction Cocktail (from Step IV: Procedures:Reagentsetupstep2)toeachwell,includingpositivecontrolandnegativecontrol.

4. Detection: Immediately, add 50µL of the 1X Detection Reagent (as diluted 1:50 in Step IV:Procedures:reagentsetupstep4)toeachwell,andreadinaluminometer.Werecommendtakingmultiplereadingsat5-minuteintervalstodeterminetheoptimalreadouttimepoint.

C.CalculationofResults:(Experimentalsample–Negativecontrol(mediaorsolvent/vehicle)PercentCytotoxicity=100X__________________________________________________________(MaximumGAPDHRelease)–(Negativecontrol(mediaorsolvent/vehicle))

aCella-TOX

0

2000

4000

6000

8000

10000

12000

14000

16000

0 2000 4000 6000 8000 10000

Ce ll / We ll

Figure:DemonstrateslinearresponseoftheaCella-TOXkit:Jurkatcellswereplatedatvariouscellconcentrationsperwell.Thelysisbufferwasaddedtoeachwell.aCella-TOXkitwasusedtodetectGAPDHenzymereleaseofthelysedcells.Datapointsshowsamplesrunintriplicatewithbackgroundsubtraction.



30

X.STOPREACTIONFORaCella-TOX:(ForHigh-Throughput/BatchProcessingapplications)

Additionalmaterialsrequired:

1. Waterbathsetat75°Ctobeabletoaccommodate4-6platesatatime,toenablebatchprocessing.

2. Platesealers(e.g.CorningCat#3095).

Procedure:

1. AftertheADCC,CMCorCDCincubationreactionat37°C,thereactionplateisequilibratedtoRTfor15minutes.

2. Add5-10μLoflysisbuffer(Part#3035)tothemaximumlysiswellsandincubatefor5minutes.(monitorlysisundermicroscope).

3. Bringtotalvolumeofthereactionto200μLbyadding100μLor125μLofADCCculturemediatoallwells.

4. Spindownplatefor1minuteat750Xg.5. Transfer 50μL of supernatant from all wells to 50μL of Enzyme assay diluent (Part #3008) in

correspondingwellsonawhiteluminescenceplate.6. Add100μLof the2XEnzymeassay reagent (Part #6001+dilutedPart #3008) containingG3P

(Part#6003)toallreactionwells.7. Sealtheplatewithplatesealer.

IncubateatRTfor15minutesinthedark.8. Placeplatesecurelyinawaterbathat75°Cfor30minutes.9. You may proceed to step 10 below or freeze plate overnight at -20°C. (with plate

sealer).10. TheplateisallowedtoequilibratetoRT.(maybeanhourorso)11. 50μLof1XDetectionreagent(Part#6002diluted1:50in1XDetectionreactiondiluent)isadded

toallwells.12. Read the plate on the luminometer (without the cover) immediately and take 2-3 time point

readings5minutesapart.Typically,theT=5(secondreading)willbetheoptimaltimepointtobeanalyzed.(thereadingjustbeforetheRLUvaluesstartdecreasing)



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Figure:TheabovegraphoverlaysthedoseresponsecurvesatE:Tof10:1generatedusingRituximabonDaudicellsandNKcellspurifiedfromdonor2271.The‘kinetic’reactionreferstothereactionrunatT=15’aftertheaCella-TOXreagentswereaddedtotheADCCreactionwellsimmediately.The‘stop’reactionreferstothereactionthatwasstoppedaftertheadditionoftheenzymeassayreagentandheatedat75°Cfor30minutesandfrozenovernight.Thedetectionreagentwasaddedjustpriortoreadingontheluminometer.IC50valueswith95%confidenceintervalsrangefrom0.002608to0.004371forthe‘kinetic’and0.001885to0.003310forthe‘stop’reaction.(4PLCusingGraphPadPrizm)

XI.References:

1. Methodsandcompositionsforcoupledluminescentassays.UnitedStatesPatent6,811,990CoreyandKinders.November2,2004

2. Coreyetal,J.ImmunolMethods,vol.207(1),Aug.22,1997,pp.43-51.3. Corey,M.J.,etal.,"AVerySensitiveCoupledLuminescentAssayforCytotoxicityandComplement-

MediatedLysis,"JournalofImmunologicalMethods207:43-51,1997.4. Crouch, S.P.M., et al., "TheUse of ATP Bioluminescence as aMeasure of Cell Proliferation and

Cytotoxicity,"JournalofImmunologicalMethods160:81-88,1993.5. Schafer, H., et al., "A Highly Sensitive Cytotoxicity Assay Based on the Release of Reporter

Enzymes, From Stably Transfected Cell Lines," Journal of Immunological Methods 204:89-98,1997.

6. Racher, LDHAssay, inCell and tissue culture: Laboratory procedures in biotechnology, A. DoyleandJ.B.Griffiths,Eds.1998,JohnWiley&Sons:Chichester,NewYork,Weinheim.p.71-5

7. Decker, T. and Lohmann-Matthes,M.L. (1988) A quick and simplemethod the quantitation oflactatedehydrogenasereleaseinmeasurementsofcellularcytotoxicityandtumornecrosisfactor(TNF)activity.J.Immunol.Meth.115,61–

8. Korzeniewski,C.andCallewaert,D.M.(1983)Anenzyme-releaseassayfornaturalcytotoxicity.J.Immunol.Meth.64,313–20.

9. NK sensitivity of Neuroblastoma cells determined by a highly sensitive coupled luminescentmethod. Henry Ogbomo, Anke Hahn, Janina Geiler, Martin Michaelis, Hans Wilhelm Doerr,JindrichCinatlJr.BiochemicalandBiophysicalResearchCommunications339(2006)pp375-379.

10. Histonedeactylase inhibitors supressnatural killer cell cytolyticactivity-HenryOgbomo,et. al -FEBSLett2007



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XII.FrequentlyAskedQuestions(F.A.Q.)PROBLEM: POSSIBLEFIX:NoluminescencesignalorlowRLUvalues CheckLuminometersettings

1.Checkluminometergainsetting.Readluminometerinstructionmanualandadjustsettingsaccordingly.2.EnsuretheproperworkingoftheinstrumentbycheckingtheRLUvalueofa1µMATPsolutionasapositivecontrol,ifrequired.ThisnumberwouldbeclosetothemaximumRLUreadingforyourluminometer.3.Areyouusingopaquewhiteplatesontheluminometer?Clearplateswillleadtothescatteringoflightandalowsignal.Pleasecontacttheinstrumentmanufacturerforrelatedtechnicalassistance.

Assay-related: 1.Uponarrival,checkifcontentsofthekitwere

storedatappropriatetemperatures.Part#’s6001,6002and6003areextremelytemperature-sensitiveandneedtobefrozenbetween-20and-40°C.

2.HavetheEnzymeAssayReagent(Part#6001)andDetectionReagent(Part#6002)gonethroughrepeatedfreeze-thawcycles?Itisrecommendedtostorethefrozencomponentsassingle-usealiquots.

3.WastheG3P(Part#6003)addedtotheEnzymeAssayReagent(Part#6001)?

4.Checkthecellsinthemaximumlysiswellsunderthemicroscopetomakesureyousee100%lysis.Ifnot,youmayhavetoperformacelltitrationwiththelysisbufferandincubationtime.

Highbackgroundissuescausingflattening ofthedose-responsecurve. 1.Areyouusinglow-IgGFBSasrecommended?

2.Havethetargetcellsbeenadaptedtothelow-IgGFBSmediaforafewpassages?

3.AreyouisolatingNKcells?UsingPBMC’scancontributetohighbackgroundduetothelargenumberofcells.

4.WasthePBMCpelletisolatedclearofRBC’sandplatelets?Acloudysupernatantmayindicatethepresenceofplatelets.Typically,awashwith1XPBS/EDTAshouldclarifythepellet.

5.Werethetargetandeffectorcellswashedandresuspendedinfreshlow-IgGFBSculture-mediajustpriortothestartoftheADCCreaction?Ifnot,thedying/deadcellswillincreasethebackgroundsignal.



33

6.DidyouaddtheG3P(Part#6003)tothe2XEnzymeAssayreagent(dilutedPart#6001)justbeforeitwasaddedtothesupernatant?

7.FollowingtheADCCreaction,istheplatebeingspundownandthesupernatantusedintheassay?Usingthelysateasawholewithoutspinningmaycontributetothehighbackgroundluminescence.

8.HavetheNKcellsbeenfreshlyisolated?Useoffrozen-and-thawedeffectorcellsintheassaymayalsocontributetoahighbackground.

Highdonor-to-donorvariation 1.UseofNKcellsratherthanPBMC’saseffector

cellsshouldminimizethisproblemtoagreatextent.2.CytokinessuchasIL-2maystimulateeffectorcellstodifferentdegreesindifferentdonors.ThismaycontributetodifferentCMCresponsesindifferentdonors,anddecreasethesignal-to-noiseratioofthedose-responsecurve.

acella-tox v1 3.1 protocol · 2020-02-14 · time-point and kinetics methods the generation of atp...

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