Project:EUNCL
LLC-PK1KidneyCytotoxicityAssay
AUTHORED BY:
DATE:
Vu To, Geir Klinkenberg
13-05-2016
REVIEWED BY:
DATE:
Matthias Roesslein
14-05-2016
Rainer Ossig
Sabrina Gioria
APPROVED BY:
DATE:
Matthias Roesslein
14-05-2016
DOCUMENTHISTORY
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Geir Klinkenberg
DocumentType
SOP
Initial Document
DocumentID
EUNCL-GTA-001
Version
1.0
Status
Page
1/15
TableofContent
1 Introduction......................................................................................................................................3
2 PrincipleoftheMethod....................................................................................................................3
3 ApplicabilityandLimitations(Scope)...............................................................................................3
4 RelatedDocuments..........................................................................................................................3
5 EquipmentandReagents..................................................................................................................4
5.1 EquipmentandCultureware.....................................................................................................4
5.2 Reagents....................................................................................................................................4
5.3 ReagentPreparation..................................................................................................................4
6 Procedure.........................................................................................................................................6
6.1 Flowchart..................................................................................................................................6
6.2 Cellhandling..............................................................................................................................6
6.3 Cellseeding................................................................................................................................7
6.4 Assayprocedure........................................................................................................................8
6.5 Calculations.............................................................................................................................10
7 QualityControl,QualityAssurance,AcceptanceCriteria...............................................................10
8 HealthandSafetyWarnings,CautionsandWasteTreatment.......................................................11
9 Abbreviations.................................................................................................................................11
10 References....................................................................................................................................12
11 Appendix......................................................................................................................................13
11.1 Cellhandlingandstorage......................................................................................................13
11.1.1 Celloriginandprovenance.............................................................................................13
11.1.2 ThawingProcedure.........................................................................................................13
11.1.3 Propagationofstockampoules......................................................................................14
11.1.4 Platemapwithpositioningofsamplesandcontrolsinassayplates.............................15
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DocumentID
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Version
1.0
Status
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1 Introduction
This protocol describes the cytotoxicity testing of nanoparticle formulations in porcine
proximal tubule cells (LLC-PK1), as part of the in vitro EU-NCL preclinical characterization
cascade. The protocol utilizes two methods for estimation of cytotoxicity, 3-(4,5-Dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and lactate dehydrogenase
(LDH)release[1-2].
2 PrincipleoftheMethod
1. MTTAssay
MTT is a yellow, water-soluble tetrazolium dye that is reduced by live cells to a water-
insoluble,purpleformazan.Theamountofformazancanbedeterminedbysolubilizingitin
DMSO and measuring it spectrophotometrically. Comparisons between the spectra of
treatedanduntreatedcellscangivearelativeestimationofcytotoxicity[3].
2. LDHAssay
LDHisacytoplasmicenzymethatisreleasedintothecytoplasmuponcelllysis.TheLDH
assay,therefore,isameasureofmembraneintegrity.ThebasisoftheLDHassay:a)LDH
oxidizes lactate to pyruvate, b) pyruvate reacts with the tetrazolium salt INT to form
formazan, and c) the water-soluble formazan dye is detected spectrophotometrically [4,
5].
3 ApplicabilityandLimitations(Scope)
TheSOPdescribesinvitromethodsforevaluationofcytotoxicityofnanoparticleformulationsusing
theMTT(metabolicactivity)andLDH-assays(membraneactivity)forLLC-PK1cells.Forbothassays,
potentialinterferencesofthenanoformulationsontheassayreadoutshouldbetakenintoaccount
duringdesignofexperimentsandinterpretationofdata.Existingavailableliteratureshouldtherefore
be reviewed during design of experiments in order to evaluate the applicability of the assays.
Theassaysprovidebasicinformationofthecytotoxicityofthenanoparticleformulations.Theassays
doesnotprovideinformationofsublethalcellulareffectsordetailedmechanisticinformationofthe
toxiceffectexperiencedbythecells.
4 RelatedDocuments
Table1:
DocumentType
SOP
DocumentID
EUNCL-GTA-001
Version
1.0
Status
Page
3/15
DocumentID
EUNCL-GTA-2
DocumentTitle
HepG2HepatocarcinomaCytotoxicityAssay
5 EquipmentandReagents
5.1
EquipmentandCultureware
1. Costar96wellflatbottomcellcultureplates(Nunc,3598)
2. Greiner96wellflatbottompolystyreneplates(Greiner,655163)
3. Halfarea96wellplate(Costar,3695)
4. Platereader(TecanInfinity200Pro-orequivalent)
5. Orbitalplateshaker
6. Incubator,37˚Cwith5%CO2and95%humidity
5.2
Reagents
1. LLC-PK1(pigkidneycells)(LGCStandards,CL-101,LOT59681631)
2. MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide)(Sigma,M5655)
3. Dimethylsulfoxide(Sigma,D5879)
4. Glycine(Sigma,G7126orG7403)
5. Sodiumchloride(Sigma,S7653)
6. 10%Triton-X-100(Sigma,93443)
7. Digitonin(Sigma,D141-500MG)
8. Medium199Cellculturemedia(Sigma,M2154)
9. Fetalbovineserum(Sigma,F7524,LOT025M3302)
10. L-Glutamine(Sigma,G7513)
11. Penicillin-Streptomycin(Gibco,15140122)
12. Dissociationreagente.gTrypsin/EDTAorTrypLE(e.gGibco,12605-010)
13. BiovisionLDH-cytotoxicityassaykit(Biovision,K311-400)
14. PBS(Difco,BR0014)
5.3
ReagentPreparation
Cellculturemedium:
ThecompletemediumforLLC-PK1shouldbemadefresheveryweekandstoredat4˚C.Ifitis
not used within a week remaining media should be discarded. The complete cell culture
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media is prepared by adding 2 mM L-Glutamine, 3% FBS and 100 U/mL PenicillinStreptomycintoMedium199.
PositiveControls:
Prepareallpositivecontrolsfreshforeachroundofassay,samecontrolcanbeusedforallreading
point(0,4,24and48hr)
1. 0.1%Triton-X-100:
Preparea20Xstock(2%)ofTriton-X-100byadding2mLofTriton-X-100to8mLofMedium
199CellCultureMedia(with3%FBS).Sterilizebyfilteringthrougha0.2µmfilter.
2. DigitoninstocksolutionismadebysolubilizingdigitonininDMSOtoafinalconcentrationat
20mg/mL.Mixuntilaclearandhomogeneoussolutionisobtained.Thestocksolutioncanbe
aliquoted and frozen at -20˚C. Prepare a 2X digitonin solution for internal plate control by
diluting digitonin stock (20 mg/mL) in Medium 199 Cell Culture Media to a final
concentrationof60µg/mL.
MTTassay:
1. MTTsolution:
Prepare5mg/mLMTTinPBS.Sterilizebyfilteringusinga0.2µmfilter.Thesolutioncanbe
storedforuptoonemonthat4˚Cinthedark,orfreezeat-20˚C.
2. GlycineBuffer:
Prepare0.1Mglycine(MW75.07)with0.1MNaCl(MW58.44)inMQwater,pH10.5.
Sterilizebyfilteringusinga0.2µmfilter.Thesolutioncanbestoredatroomtemperaturefor
upto4weeks.
LDHassay:
1. Reconstitute catalyst in 1 mL dH2O for 10 min with occasional vortexing. The solution is
stablefor2weeksat4˚C.
2. Reaction mixture (for one 96-well plate): Add 250 µL of reconstituted catalyst solution to
11.25mLofdyesolution.Oncethawed,thekitcomponentsarestablefor2weeksstoredat
4˚C.Reconstitutedcatalystsolutionshouldbeaddedtothedyesolutionimmediatelybefore
use.
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6 Procedure
6.1
Flowchart
Figure1:Briefoutlineoftheworkflow.
6.2
•
Cellhandling
The LLC-PK1 cell line used by EUNCL is obtained from LGC Standards, see Annex for detailed
informationaboutthawingandpropagationofasetofstockampoules.
•
Thesestockampoulesserveasstartingpointforallexperiments.
•
After thawing of stock ampoules, cells are subcultured for 3 passages or more until enough
amountofcellsareachieved(seeSubcultivation).
•
Onthedayofexperiment,performcellseedingasdescribedin6.4,continuewithadditionof
samplesthefollowingdayasdescribedin6.5.
Subcultivation
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LLC-PK1 cells are kept in a sub confluent state by routinely passaging twice or three times a
weektoseedingdensitiesbetween1.2x104–3.2x104cells/cm2.
Givenvolumesarefor75cm2flask–proportionallyreduceorincreaseamountofdissociation
mediumforculturevesselofothersize.
1. Removeanddiscardculturemedium.
2. Wash the cell layer twice by gently rinsing it with 10-15 mL preheated (37˚C)
Dulbecco'sphosphate-bufferedsalinewithoutcalciumandmagnesium.
3. Add 2.0-3.0 mL dissociation reagent (e.g Trypsin/EDTA or TrypLE®), incubate at 37˚C
for10minutes,andgentlyknockcultureflaskstodetachmostofthecells.
4. ResuspendcellsinmediumcontainingFBStostoptrypsination.
5. Transferanddiluteinnewculturevessels.
6. Incubate the culture at 37˚C in a humidified atmosphere with 5% CO2 in a suitable
incubator.
6.3
Cellseeding
Cellpreparation(orasrecommendedbysupplier,seealso6.3.)
1. Harvest cells from prepared flasks, the cells should be cultivated for minimum 3 passages
before use for experiment. Note: Limit to 20 passages from vial from cell bank at LGC
Standards(Figure3).
2. Countcellnumberusingacoultercounterorhemocytometer.
5
3. Dilutecellstoadensityof2.5x10 cells/mLinMedium199(3%FBS)cellculturemedia.
4. Plate100µLcells/wellasperplateformat(Annex)forfour96-wellplates(timezero,4,24,
and48hrsampleexposure).Seeplatedesign.
5. Incubate plates for 24 hr at 5% CO2, 37˚C and 95% humidity. Cells are grown to
approximately80%confluence(Figure2).
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Figure2.LLC-PK1CellCulture.Imagewastakenwithaphasecontrastmicroscopeat
225Xmagnification.LLC-PK1cellsareapproximately80%confluentatthisstage.
6.4
Assayprocedure
The cells are prepared in 96-well plates as described in section 6.4. See also plate design in
Annex. The format indicates no cells in rows A and H as they serve as particle blanks to be
subtractedfromcelltreatmentwells.Eachplateaccommodatestwosamples(RowsA–DandEH).Eachnanoparticleistestedatninedilutions.Column11receivestheTritonX-100attheend
oftherelevanttimepointandcolumn12receivesthepositivecontrolDigitonin.
TimeZeroPlate(MTTAssay)
1. Removetimezeroplatefromtheincubatorandadd100µLfreshculturemediatoallwells.
Thenadd10µLof20XTriton-X-100tothepositivecontrolwellsincolumn11(seeplate
format in Annex) for a final concentration of 0.1% Triton-X-100 (see Section 5.3). Let the
platesetfor10minutesatroomtemperature.
2. Remove100µLofmediafromeachwellandtransferittoanotherplate(Greiner,
655163), maintaining plate format. Transfer 50 µL if half area plates are used for
LDHassay(Costar,3695).UsethisplateimmediatelyfortheLDHassay(seeSection
below).
3. Removeremainingmediafromoriginalplateanddiscard.
4. Add200µLfreshmediatoallwells.
5. Add50µLMTT(seeSection5.3)toallwells.
6. Coverwithaluminumfoilandincubateat37˚Cfor3-4hrs.
7. Aspirateanddiscardmedia.
8. Add200µLDMSOtoallwellstosolubilizetheMTTformazancrystals.
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9. Add25µLglycinebuffer(seeSection5.3)toallwells.Coverwithaluminumfoilandplaceon
shakertomixfor10minutesinroomtemperature.
10. Readabsorbanceat570nmonplatereaderusingareferencewavelengthof680nm.
TestSamplesandPositiveControlAddition
Allsamplesarerunintriplicateswith9dilutionseach(seePlateMap).
1. The highest concentration of nanoparticle tested should be at the limit of solubility or
determinedbythepotencyofthetestmaterial.
2. Dilutethetestcompoundinmedia,makingatotalofnine1:4dilutionsat2Xofthedesired
final concentration. Different dilution series (i.e. 1:2, 1:10) can be done depending on test
materialpotencyandavailability.
3. Add100µLofeachsampledilutionandpositivecontrolto4,24and48hrexposureplatesas
pertheplateformat(Annex),andplacein37˚Cincubatorwith5%CO2and95%humidityfor
indicatedtime.NotethatTritonX-100controlisaddedlaterattheendoftheexposuretime,
so that these wells receive only 100µL media at this point (See step 1 in MTT assay).
Alternatively,sampleandpositivecontrolcanbemadeatdesiredfinalconcentration.Media
isaspiratedfromthewellsonthetestplates,andsampleandpositivecontrolareaddedat
200μLperwellaspertheplateformat(Annex).
TestPlates,4,24and48hrexposures(MTTAssay)
1. Attheendofeachexposuretimepoint,removeplatefromincubatorandadd10µLof
20XTriton-X-100topositivecontrolwells(seeplateformatinAnnex)forafinal
concentrationof0.1%Triton-X-100(seeSection5.3).Lettheplatesetfor10minutesat
roomtemperature.
2. Remove100µLofmediafromeachwellandtransferittoanotherplate(Greiner,
655163),maintainingplateformat.Transfer50µLifhalfareaplatesareusedforLDH
assay(Costar,3695).UsethisplateimmediatelyfortheLDHassay(seeSectionbelow).
3. Removeremainingmediafromoriginalplateanddiscard.
4. Add200µLfreshmediatoallwells.
5. Add50µLMTTtoallwells.
6. Coverwithaluminumfoilandincubatefor37˚Cfor3-4hr.
7. Aspirateanddiscardmedia.
8. Add200µLofDMSOtoeachwell.
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9. Add25µLofglycinebuffertoeachwell.Coverwithaluminumfoilandplaceonshaker
tomixfor10minutesinroomtemperature.
10. Readabsorbanceat570nmonplatereaderusingareferencewavelengthof680nm.
Testplates,0,4,24and48hrexposures(LDHAssay)
(AdaptedfromBiovisionLDHCytotoxicityAssayKit,K311-400)
1. Add100µLoftheReactionMixture(seeSection5.3)toeachwelloftransferplate.
Alternativelyadd50µLoftheReactionMixtureifhalfareaplatesareused.Shakeplate
onanorbitalshakerbrieflytomixsamples.
2. Incubateatroomtemperatureforupto20minutesinthedark.
3. Readtheplateonaplatereaderat490nmusingareferencewavelengthof680nm.
6.5
Calculations
Allsamples,positive,negative,andmediacontrolsarerunintriplicate(e.g.,rowsB-DorE-G).Each
wellwillbesubtractedfromitsrespectivecell-freeblank(e.g.,B2-A2orG3-H3)inthefollowing
calculations.Theaverageofthesethreevaluesshouldbeusedintheequationsbelowforthe
positiveandnegativecontrols(e.g.,[(B10-A10)+(C10-A10)+(D10-A10)]/3=meanmediacontrol
absorbanceforsample1,or[(B11-A11)+(C11-A11)+(D11-A11)]/3=meanTriton-X-100positive
controlabsorbanceforsample1.Sample2usestheblankrowHforsubtractions.).
1.
MTTAssay
% Cell Viability =
2.
sample absorbance − cell free sample blank
mean media control absorbance
×100
LDHAssay
% Total LDH Leakage =
!"#$%& !"#$%"!&'(!!"## !"## !"#$%& !"#$% !!"#$ !"#$% !"#$%"& !"#$%"!&'(
!!"! !"#$%&' !"#$%$&' !!"#!!" !!"#!!!!!!!!"#$ !"#$% !"#$%"& !"#$%"!&'(
×100
3.
Mean,SDand%CVshouldalsobecalculatedforeachpositivecontrol,negativecontroland
unknownsample.
7 QualityControl,QualityAssurance,AcceptanceCriteria
Acceptancecriteria
1. Asetofassaywellsexposedtoadilutionseries(9dilutions,3parallelwellsper
concentration)ofthepositivecontroldigitoninfor24hoursisincludedineach
assaybatch,startingat140µg/ml.TheEC50valueofthedigitoninpositivecontrol
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isusedformonitoringofassayperformance.Plotvaluesforeachassaybatch.
AssaysbatchesinwhichtheEC50valueofthedigitoninpositivecontrolare
outsideof3standarddeviationsshouldbediscarded.
2. Theviabilityinwellswiththeplateinternaldigitionincontrolshouldbelessthan
50%.
3. Thereplicatecoefficientofvariationsofthepositivecontrolsandofthesamples
shouldbewithin50%.
4. Theassayisacceptableifcriteria1and3aremet.Otherwise,theassayshouldbe
repeateduntilacceptancecriteriaaremet.
5. Iftheacceptancecriteriaaremet,determinethehighestconcentrationofthe
nanoparticulatematerialthatdoesnotinterferewiththeassaysystemindicated
inrowsAandH.
6. Theconcentration–responsecurvesforthe48hrMTTandLDHdatashouldbe
classifiedashavingcomplete(twoobservedasymptotes)orincomplete(second
asymptotenotobtained)curves,singlepointactivity(activityatthehighest
concentrationonly),ornoactivity.Forallcomplete48hrconcentration–response
curves,anonlinearfitofthesigmoidalHillequationshouldbeperformed,andan
estimateofpotency(EC50-CalculatedusingGraphpadPrism),efficacy(Emax),
minimumresponse(E0),andHillslope(γ)fromtheHillequation(below)fitshould
bereported.Anyexcludedpoints(excludedbyoutlieranalysis)shouldalsobe
reported.
E=E0 +[(Emax –E0)Cγ/ECγ50 ·Cγ]
8 HealthandSafetyWarnings,CautionsandWasteTreatment
PleaserefertoavailableH.S.Einformationforanynanoformulationsevaluatedintheassays.Note
thatsomeofthelistedreagentsarehazardousandmustbehandledwithprecaution.Pleasereferto
safety data sheets for each reagent, wear protective equipment, and dispose waste according to
localregulations.
9 Abbreviations
CV
coefficientofvariation
DMSO
dimethylsulfoxide
FBS
fetalbovineserum
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INT
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazoliumchloride
LDH
lactatedehydrogenase
LLC-PK1
renalepithelialcellline,porcinekidney
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
MW
molecularweight
NaCl
SodiumChloride
PBS
phosphatebufferedsaline
SD
standarddeviation
10 References
1. ISO10993-5,Biologicalevaluationofmedicaldevices:Part5,Testsforinvitro
cytotoxicity.
2. F1903–98,StandardPracticeforTestingforBiologicalResponsestoParticlesinvitro.
3. Alley,etal.(1988)CancerRes.48:589-601.
4. Decker,T.&Lohmann-Matthes,M.L.(1988)J.ImmunolMethods15:61-69.
5. Korzeniewski,C.&Callewaert,D.M.(1983)J.ImmunolMethods64:313-320.
6. Freshney,R.I.(2010).Cultureofanimalcells:amanualofbasictechniqueandspecialized
applications.(pp.87-88,193)Hoboken,N.J.,Wiley-Blackwell
7. NCLMethodGTA-1,Version1.2,November2015
LGCStandards
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11 Appendix
11.1 Cellhandlingandstorage
11.1.1 Celloriginandprovenance
LLC-PK1porcineproximaltubulecellsareusedasafunctionalmodelinthekidneycytotoxicity
assayandmustbewellcharacterizedandvalidated.ValidatedcelllinesareobtainedfromLGC
Standards. Upon arrival the LLC-PK1 cells should be propagated and cryopreserved at a low
passagenumber.InordertominimizevariationsbetweenEU-NCLlabsduetocellcultivation,
celllinesusedintheEU-NCL-GTA01assayshouldnotbepropagatedabove20passagesfrom
originalvialsfromLGCStandards(Seefigure3)[6,7].
Figure3:PropagationofstockampoulesfromLGCStandards.
11.1.2 ThawingProcedure
Thawingoffrozenampoulewasdoneaccordingtoinstructionsfromcellbank[8].
1. Thawthevialbygentleagitationina37˚Cwaterbath.Toreducethepossibilityof
contamination,keeptheO-ringandcapoutofthewater.Thawingshouldberapid,
approximately1-2minutes.
2. Removethevialfromthewaterbathassoonasthecontentisthawed,anddecontaminate
bysprayingwith70%ethanol.Placethevialintobiosafetycabinetandworkunderstrict
asepticconditions.
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3. Transferthevialcontenttoasterile15mLconicalcentrifugetubecontaining9.0mL
preheatedcompleteculturemedium,andspinat140xGfor5minutes.Discardsupernatant.
4. Resuspendthecellpelletgentlyin2-3mLofthecompletecellmedium(thissmallvolume
makesiteasiertoachieveahomogeneoussuspension).Gentlydilutethecellsuspensionto
therecommendedfinalvolumeinculturevessel.
5. Incubatethecultureat37˚Cinahumidifiedatmospherewith5%CO2inasuitableincubator.
11.1.3 Propagationofstockampoules
Stock ampoules were propagated by thawing vial from cell bank according to thawing procedure
(11.2.2). For LLC-PK1 the vial from cell bank was thawed, old media containing freeze agent was
removed by diluting in fresh media before spinning down the cells. Cell pellet was resuspended in
freshmediaandtransferredtoaT25culturevessel.Thecelllinebecame70-80%confluentaftertwo
days,andwaspassagedtwicebeforefreezingofstockampoules.
P1:Thawto10mLinT25(2days)
P2:Splitandseedto2.6x104cells/cm2toT75(1-2days)
P3:Splitandseedto1.2x104cells/cm2(3days),freezestockampouleswhencellsbecome70-80%
confluent.
FreezingProcedure
Alwaysstartbypreparingallreagents,vialsandequipmentneededforfreezingcells.Afterthecells
areoutoftheincubator,minimaltimeshouldbeusedforsuchpreparationsteps.
1. Harvestcellsaccordingtothedescriptionforsubculturing.Afterdissociationofcells,dilute
theminfreshculturemedium.
2. Measurecelldensityandviabilitybytakingoutanaliquotofthecellsuspension.Calculate
thenecessaryvolumeoffreezingmedia(SeeFreezingMediaunderneath).
3. Transfercellsuspensiontocentrifugetubes,andspindowncellsat140xGfor5-7minutes.
Avoidverydensecellsuspensionsasthesemightbedifficulttospindown.
4. Removeallmedium.
5. Gently resuspend cells to wanted concentration in fresh freezing medium. Freeze LLC-PK1
cellsat1x106–2x106cells/mLincryogenicvials,aliquot1mLineachvial.
6. Transfercryovialstofreezingcontainers(CoolCell®),andplacethecontainerin-80˚Cfreezer,
leaveforatleastfourhoursbeforetransferringtostorageinliquidnitrogen.
FreezingMedia
1. ToprepareLLC-PK1freezingmediausecompletesupplementedmediumandaddsterile
DMSO(DimethylsulfoxideHybri-MaxTM(Sigma,D2650orequivalent)toafinalvolumeof5%
(v/v).
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11.1.4 Platemapwithpositioningofsamplesandcontrolsinassayplates
1
Media
A TS1,D9
B
C
D
E
F
G
H
2
Media
TS1,D8
3
Media
TS1,D7
4
Media
TS1,D6
5
Media
TS1,D5
6
Media
TS1,D4
7
Media
TS1,D3
8
Media
TS1,D2
9
Media
TS1,D1
10
Media
11
Media
Triton-X
0.1%
Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells
Cells
TS1,D9 TS1,D8 TS1,D7 TS1,D6 TS1,D5 TS1,D4 TS1,D3 TS1,D2 TS1,D1 Media
Triton-X
0.1%
Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells
Cells
TS1,D9 TS1,D8 TS1,D7 TS1,D6 TS1,D5 TS1,D4 TS1,D3 TS1,D2 TS1,D1 Media
Triton-X
0.1%
Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells
Cells
TS1,D9 TS1,D8 TS1,D7 TS1,D6 TS1,D5 TS1,D4 TS1,D3 TS1,D2 TS1,D1 Media
Triton-X
0.1%
Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells
TS2,D9 TS2,D8 TS2,D7 TS2,D6 TS2,D5 TS2,D4 TS2,D3 TS2,D2 TS2,D1 Media
Triton-X
0.1%
Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells
TS2,D9 TS2,D8 TS2,D7 TS2,D6 TS2,D5 TS2,D4 TS2,D3 TS2,D2 TS2,D1 Media
Triton-X
0.1%
Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells
TS2,D9 TS2,D8 TS2,D7 TS2,D6 TS2,D5 TS2,D4 TS2,D3 TS2,D2 TS2,D1 Media
Triton-X
0.1%
Media Media Media Media Media Media Media Media Media Media
Media
TS2,D9 TS2,D8 TS2,D7 TS2,D6 TS2,D5 TS2,D4 TS2,D3 TS2,D2 TS2,D1
Triton
0.1%
12
Media
DIGI
30µg/mL
Cells
DIGI
30µg/mL
Cells
DIGI
30µg/mL
Cells
DIGI
30µg/mL
Cells
DIGI
30µg/mL
Cells
DIGI
30µg/mL
Cells
DIGI
30µg/mL
Media
DIGI
30µg/mL
Abbreviations:
TSX:TestsamplenumberX
DX:DilutionnumberX
DIGI:Digitoninplateinternalcontrolsample
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