cell counting, viability & proliferation

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Cell counting, viability & proliferation

Technical tipCellcountingisrequired» To monitor cells during cell cultures» For cell preparation or any cell experiment» To standardize cell samples for analysis. » Cellproliferation» Cytotoxicityassays

Severalmethods have been proposed, each fittingmore or less to each specificapplication:countingdeadcellsmaybeacceptableforthepreparationofcellextractsordesiredwhenonedonotwanttooperatewithhazardouscellsorforcytotoxicitystudy.Attheoppositedeadcellscountingisgenerallyprecludedforcellcultureandbioassays.Itmaybeusefultoquantitateonlyviablecells,oronlyfastproliferatingcells.

Interchimprovidesalargechoiceofcellassayscoveringstandardaswellasinnovativemethodsforgeneraltospecificcellassays.

SelectionguideProbe Principle Detection Method Dead Viable Proliferating Features/Advantages-DrawbacksTrypanblue Membraneexclusion Colorimetric ++ ++ ++ Cheap,buttimeconsuming,notscalable.

Microscopy Donotstateonviability.

Hoechst DNAprobeexclusion Fluorimetric ++ ++ +++ Cheap,Scalable,Nontoxic.Donotstateonviability.MorerapidthanMTT/XTT;unfixedorfixedsamples.

MTT Formazan dye, orange precipitate. Colorimetric - ++ +++

Popularmethod.Sensitive,Scalable.NontoxicIncreasedsolubilityandperformancefromMTTtoXTTandWST.

XTT SameasMTTbutmoresoluble. Colorimetric - ++ +++

WST Formazandye,soluble&not toxic - ++ +++

UptiBlue ratiometricblueprobe Colorimetric - +++ +++ Nosolubilizationstep(unlikeMTT).Applyalsotofor cell redox Fluorimetric adherentcells.SensitivitysimilartoMTT/XTT,but

easiertouseFluorimetry/SuperiorsensitivitytoMTT/XTT.

Calcein-AM Calceinaccumulationin Fluorimetric - +++ ++ Nosolubilizationstep(unlikeMTT/XTT).Adaptabletoacytoplasm widevarietyoftechniques,including:microplateassays,

invivocelltracing.Donotworkforbacteria.Mayaltersomecellfunctions.

GAPDH Release ofGAPDH coupledtoATPassay Bioluminescence - +++ + Measurement of Cell-Mediated (TCells,ADCC,NK) or

Complement-MediatedCytolsis.

CFSE Fluoresceinproteinlabeling Fluorimetric ++ ++ ++ Usefulwhenothermethoddonotworkproperly.Donotstateonviability.

AnnexinV AnnexinV/PhosphoSerine Fluorimetric + +++ + UsefulforApoptosisstudy.

LDH convertion in colored product - ++ + RecommendedforcytotoxicityassaysSerumInterference.

LuciferinSyst. ATPmeasure Luminescence - + +++ Pros:sensitivity/linearity.Cons:signaldependsoneachcellline,ontemperature

-3HThymidine DNAincorporationof Radioactivity - + +++ Cons:hazardous(radioelements).radioactivity

BRDU DNAincorporation Immunoassay - + +++51Cr release EU3+ Releaseofradioactivityby Radioactivity - - +++ Recommendedforcytotoxicityassays.

cytoplasm Cons:hazardous(radioelements).

Propidium 7-Membranepermeability Fluorimetric +++ - - Usedincombinaisonofgreenfluorescencedyelike.Iodide,AAD AnnexinV-FP488todiscriminatedeadcellsfromalivecells.

MicroPlatereaders&ImagingsystemsInterchimandBertholdcollaborationsupports furtheryourworks.Manyofourfluorescenceandluminescencereagentsandkitswerevalidatedwithinstruments.

*NightOWLLB983NC100

*MithrasLB940MultiModeReader

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Viability &

Cytotoxicity A

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Dual staining to detect live and dead cells

■Live/DeadMammalianViabilityAssayKitTwo-colorfluorescentstainingoflive(green)anddeadcells(red)

» DualDetection:Detectbothliveanddeadcellssimultaneously.» Simple&Fast:Requireonlya30-mindyeloadingtimeandthenmeasurewithoutwashing.» Economical:Performviabilityandcytotoxicityassaysatthesametime.» Versatile:Analyzewithflowcytometers,fluorescencemicroscopesorfluorescenceplatereaders.

TheViability/CytotoxicityAssayKitforLive/DeadCellsprovidesatwo-colorfluorescencestainingonbothliveanddeadcellsusingtwoprobesthatmeasuretworecognizedparametersofcellviability—intracellularesteraseactivityandplasmamembraneintegrity[Papadopoulos,1994].Thekitissuitableforusewithfluorescencemicroscopes,fluorescencemultiwellplatescannersandflowcytometersandotherfluorescencedetectionsystems.Theassayprinciplesaregeneralandapplicabletomosteukaryoticcelltypes,includingadherentcells[Vaughan,1995]andcertaintissues[Poole,1993],butnottobacteriaoryeast.Thisfluorescence-basedmethodofassessingcellviabilitycanbeusedinplaceoftrypanblueexclusion,51Cr releaseandsimilarmethods for determining cell viability andcytotoxicity. It is generally faster, lessexpensive, saferandamore sensitiveindicatorofcytotoxiceventsthanalternativemethods.EthD-IIIsharesthesamepropertywithEthD-IusedinLive/DeadViability/CytotoxicityAssayKit#486301andis40%brighteratintensitycomparedtoEthD-I.ValidityoftheLive/DeadViability/Cytotoxicityassayforanimalcellapplicationshasbeenestablishedbyseveralpublications.

Livecellsaredistinguishedby thepresenceofubiquitous intracellularesteraseactivity,determinedby theenzymaticconversionof thevirtuallynonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells,producing an intense uniform green fluorescence in live cells (ex/em ~495 nm/~515 nm). EthD-III enters cells with damagedmembranes andundergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em~495nm/~635nm).EthD-IIIisexcludedbytheintactplasmamembraneoflivecells.Thedeterminationofcellviabilitydependsonthesephysicalandbiochemicalpropertiesofcells.Cytotoxicevents thatdonotaffect thesecellpropertiesmaynotbeaccuratelyassessedusingthismethod.Backgroundfluorescencelevelsareinherentlylowwiththisassaytechniquebecausethedyesarevirtuallynonfluorescentbeforeinteractingwithcells.

Ifcellsarefirstfixed,andthenstained,theLive/DeadBacterialViability/Cytotoxicitykitcanalsobeconsidered.ToreplacethedyeCalceinAMthatwillonlystainthelivecells,theDMAO;aDNA-bindingdye,willstainbothintactanddamagedcellmembranes.

References:JImmunolMethods,177,101(1994).JCellSci,106,685(1993).JNeurosci,15,5389(1995).

Description P/N : QtyLive/DeadMammalianViabilityAssayKit FP-BF4710 1000 tests in microplate reader

Relatedproducts:DMAO,nucleistainforlivecells,2mMsolninDMSO FP-CA8150 1 mlEthidiumBromideIII,1mMsolution FP-BP9341 200µlMTT(λabs.(cleaved):650nm(550-600nm)) FP-65939A 1 gLive/DeadYeastViabilityAssayKit 486301 1kitbasedoncalcein-AMandPI. HelaCellsincubatedwithassaysolution.

■Live/DeadBacterialViability/CytotoxicitykitTwocolorfluorescencestainingonbothlivebacteria(green)anddeadbacteria(red)

» DualDetection:Detectliveand deadbacteriacellsinacell population simultaneously. » Simple&Fast:15mindyeloading andmeasurewithoutwashing.» Economic:Performviabilityand cytotoxicity assays at the same time. » Versatile:Analysiscompatiable withflowcytometersand fluorescencemicroscopesusing popularsettingsforfluorescein and propidium iodide.

Viability/CytotoxicityAssayKit forBacteriaLive&DeadCellStainingKitprovides two-colorfluorescencestainingonboth live (green)anddead(red)bacteriausingtwoprobes,DMAOandEtD-III.DMAOisagreen-fluorescentnucleicaciddyethatstainsbothliveanddeadbacteriawithintactanddamagedcellmembranes.EtD-IIIisared-fluorescentnucleicaciddyethatstainsonlydeadbacteriawithdamagedcellmembranes.WithanappropriatemixtureofDMAOandEtD-III,bacteriawith intactcellmembranes isstainedfluorescentgreen,whereasbacteriawithdamagedcellmembranesisstainedfluorescentred.Thekitissuitableforusewithfluorescencemicroscopesandflowcytometers.Theassayprinciplesaregeneralandapplicabletomostbacteriatypes.

Acommoncriterionforbacterialviabilityistheabilityofabacteriumtoreproduceinsuitablenutrientmediathatisreferredtoasgrowthassays.Thiskityieldsresultsthatcorrelatewellwithgrowthassaysinliquidorsolidmedia.Undercertainconditions,however,bacteriahavingdamagedmembranesmaybeabletorecoverandreproduce—suchbacteriamaybescoredas"dead"inthisassay.Conversely,somebacteriawithintactmembranesmaybeunabletoreproduceinnutrientmedium,andyetthesemaybescoredas"alive".Therefore,thesesituationsneedtobeconsideredifavastdifferenceofliveanddeadbacteriacountsisobservedbetweenthisassayandgrowthassays.

Thiskitcanalsobeconsideredifcellslikemammaliancells,arefirstfixed,andthenstained.

Description P/N : QtyLive/DeadBacterialViability/Cytotoxicity FP-BU1040 1000 tests in microplate reader

Relatedproducts:Live/DeadYeastViabilityAssayKit 486301 1KitbasedonWST-8formazandye.Readat450nm(450-490nm)

Viability &

Cytotoxicity A

■Ethidiummonoazide,bromide(EMA)Selectively and covalently labels membrane-damaged or metabolicallycompromised cells in the presence of live cells

Ethidium monoazide bromide is a red fluorescent nucleic acid stain with aphotoaffinity label.The dye, after photolysis, binds covalently to nucleic acids.1 AfterphotocrosslinkingtoDNA,thewavelengths(λexc. /λem.=504/600nm)arecompatiblewithasimultaneousobservationofanothergreen indicator.Thedyehasbeenusedto"footprint"drugbindingsitesonDNA2tomodifyplasmidDNA,3,4 and to determine hemopoietic cell phenotype, function and position in the cell cycle.5Aparticularlyusefulapplicationofthedyeistoselectivelyandcovalentlylabeldeadcellsinthepresenceoflivecells.Sinceethidiummonoazidebromideis relatively impermeant to livecells, itselectively labelsDNA indeadcells inamixedpopulationof liveanddeadcells.Photolysisfollowingthedyeapplicationrenders thedeadcellDNAcovalently labeledwith thedye.Onecan thenwashandfixthecellpreparationandexamitbymicroscopyfluorescenceplatereaderor flowcytometry.Themajor advantageof thismethod is that researchers canavoid extensive manipulation of live pathogenic organisms.6At thedifferenceofpropidiumiodide,theethidiummonoazidebindscovalently,and,whenappliedtocellsbeforefixation,providesanindicationofwhatfractionoftheunfixedpopulationweremembrane-damagedormetabolicallycompromised.

References:1)J.Mol.Biol.92,319(1975)2)Euro.J.Biochem.182,437(1989)3)J.Biol.Chem.257,13205(1982)4)J.Biol.Chem.259,11090(1984)5)Cytometry11,610(1990)6)Cytometry,12,133(1991)7)PNAS,97,no.17,p.9504-9507(2000)

TwophotonfluorescentimageoflivePTK2cellsvitallystainedwithEMA:Alateprometaphasecellillustratingthehighselectivityofthestainforthechromosomes.

Description P/N : QtyEthidiummonoazide,bromide(EMA) FP-48256A 5mgλex./λem.(DNAbound):504/600nm;MW:420.3

■Propidiummonoazide(PMA)Selectivelyandcovalentlylabelsdeadcellsinthepresenceoflivecells

PMATMisaderivativeofEMA,butithassignificantlyhigherDNAbindingaffinityandiscellimpermeant.AsEMA,afterphotolysis,thedyeisconvertedtoafluorescentDNAstaincovalentlyboundtoDNA.

Description P/N : QtyPropidiummonoazide(PMA) FP-BZ9340 1 mgλex./λem.(DNAbound):510/610nm;MW:512

References:Nocker,A. et al.,Comparisonofpropidiummonoazidewithethidiummonoazidefordifferentiationoflivevs.deadbacteriabyselectiveremovalofDNAfromdeadcells.J.Microbio Meth. 67(2),310-320(2006).Nocker A. et al., UseofPropidiumMonoazideforLive/DeadDistinctioninMicrobialEcology,Applied and Environmental Microbiology,p.5111-5117,Vol.73,No.16(2007).PanY.,BreidtF., Enumeration of Listeria monocytogenesbyReal-TimePCRwithPropidiumMonoazideandEthidiumMonoazideinthePresenceofDeadCells,Appl. Environ. Microbiol.doi:10.1128/AEM.01198-07(2007).

Live cell staining

Technical tipCalcein dye is a polyanionic derivate of fluorescein thatexhibits fluorescence that is essentially independentof pH between 6.5 and 12. The excitation and theemissionwavelengthsofcalceinare485nmand535nm,respectively.Itiswellretainedincells.Thesefeatureshavemade it a popular and versatile dye for various applications, including cell volume changes in neurons and other cells, endocytosis, gap junctional communication, membraneintegrityandpermeability,angiography,liposomes…It is worthy to notice that calcein is strongly quenchedby several ions, including Fe3+, Co2+, Cu2+ and Mn2+ at physiological pH (not by Ca2+ or Mg2+ ions). Ions levelsshouldthusbemonitored.

AM ester is membrane-permeant and enters readily cellmembranes. Intracellularesterasesconvert it intocalcein.The DMSO solution is more convenient (time saving,reduces solubilization variability) especially for morereproductiblescreeningassays. Fluorescence of calcein at pH9.0

■CalceinAMCellCounting&ViabilityAssayKitTheCalcein-AMKitprovidesasimple, rapidandaccuratemethod tomeasurecellviabilityand/orcytotoxicity.ThekitutilizescalceinAMfor thefluorometricdeterminationoflivingcellnumbers.Theamountofafluorescentdyereadat512nm,calcein,hydrolyzedbyesterasesincells,isdirectlyproportionaltothenumberofviablecellsinculturemedia.The96-wellmicroplateassayhasadetectionrangeoflessthan50cellstomorethan25000cellsperwell.Itcanbeusedfor384-wellplatesbyadding5µl(insteadof10µl)assaysolutionto50µlPBSsolutionperwell.Sinceesterasesandphenolredintheculturemediuminterferewiththefluorescencemeasurement,replacingthecellculturemediumwithPBSisnecessarypriortoaddingtheCalcein-AMassaysolution.Anincubationof10to30minutesgivessufficientfluorescenceintensityforthecellviabilitydetermination.

Features:» Suitableforproliferatingandnon-proliferatingcells» Idealforbothsuspensionandadherentcells» Non-radioactivemicroplate» Rapid(nosolubilizationstepasinanMTTassay)» Idealforhigh-throughputassays» Betterretentionandbrightnesscomparedtootherfluorescentcompounds(i.e.fluorescein)

Applications :» Celladhesion,chemotaxis,multidrugresistance,cellviability,apoptosis,cytotoxicity,...» Microplateassays,immunocytochemistry,flowcytometry,andinvivocelltracing

Description P/N : QtyCellCountingKit,calcein-AMbased 876981 500tests

876982 2x500tests

CalceinAM FP-895514 1 mgλex./λem.(cleaved):494/517nm;MW:994.9 FP-895515 20x50µg

CalceinAM,1mg/mlinanhydrousDMSO FP-855422 1 mlCalceinAM,4mg/mlinanhydrousDMSO FP-FI9820 100µlCalceinAM,5mMinanhydrousDMSO,PureGrade FP-JQ8140 200µl

Relatedproducts:AnnexinV-FluoProbes488,FlowCytometryGrade(495/519mm) FP-BH9390 100 testsPropidiumiodide,1mg/ml FP-36774A 10 ml

Viability &

Cytotoxicity A

Live cell staining

■CFDA,SEformicrobialandcellenumeration5-(and6-)-carboxyfluoresceindiacetate,succinimidylester(CFDA,SE)efficientlystainsgram-negativeandgram-positivebacterialgenerawithoutcausingundesirableeffectsoncelladhesionorviability.Thehighthroughputmethodusingmicroplatespectrofluorometryhasadetectionlimitofmid-105CFDA-stainedcells/ml.CFDA,SEtrackingtechniquehasapplicationsinbacterialtransport,publichealthmicrobiology,allowingthemovementofpathogentobemonitoredinterrestrial,aquatic,andevenfood-processingenvironments.Thetechniquemayalsobeusefulforstudyinginfectionandcolonizationbypathogensin vivo using animal models.

Reference:MarkF.etal. -DevelopmentofaVitalFluorescentStainingMethodforMonitoringBacterialTransport inSubsurfaceEnvironments,AppliedandEnvironmentalMicrobiology,October2000,p.4486-4496,Vol.66,No.10

Description P/N : QtyCFDA-SE(CFSE,GreenCellTrackingreagent) FP-52493A 25mgλex./em.(cleaved):495/519nm;MW:557

■UptiBlueCellViabilityAssayKitSubstrate:Resazurinλex./em.:540/590nmSensitive:100cells

Description P/N : QtyUptiBlueCellViabilityAssayKit UP669412 25ml

UP669413 100 ml

Applications

Cellproliferationassay

Detection of cell Growth of 4 Cell Lines usingUptiBlue

Kinetic/longtermassays

Kinetic reduction curvewithUptiBluewith platingdensityfrom500to10000cellsA549/ml.

Cytotoxicity assay

DeterminationofDoxorubicinLD50usingUptiBlueandXTT

Principle:theUptiBluedyeentersreadilyintocells,whereitelicitsawavelengthshiftofabsorbanceandastrongfluorescencerelatedtoredoxpotentialincell,informingoncellenergeticstate.

UptiBlueshowsexcellentcorrelationtoformazanandtritiatedthymidinetechniques,whilebeingmucheasierandsafertouse.ItespeciallyreplacesadvantagelyMTT/XTTinmanyapplications,fromcellcountingtoproliferationassayandcytotoxicitytesting.Furthermoreitallowslongerstudies.

Live cell staining

■WST-8CellProliferationandCytotoxicityAssayKit» Colorimetricmicroplateassay» Ready-to-useone-bottlesolution» Safe : no radioisotope or organic solvent required» No toxicity to cells» Easyandfast:noharvesting,washingorsolubilizationsteprequired» MoresensitivethanMTT,XTT,MTSandWST-1

Reducedtoxicityofassaysolution:

CCK-8consistsofWST-8and1-methoxyPMSasanelectronmediator.Aftertheplateisincubatedfor1-4hoursintheincubator,theabsorbanceismeasuredin96or384-wellplate.Thewavelengthrangeforthemeasurementoftheabsorbanceisbetween450nmand490nm.Theamountoftheyellowcoloredformazandyegeneratedbydehydrogenasesincellsisdirectlyproportionaltothenumberofviablecellsinaculturemedium.ThesensitivityusingCCK-8ishigherthanthatusingMTTortheothertetrazoliumsaltsthatproducewater-solubleformazandyessuchasXTTorMTSforHeLacellsandHL60cells.Furthermore,thecellproliferationassaydatausingCCK-8correlateswiththatusingthe3H-thymidine incorporate assay.

Description P/N : QtyCKK-8CellCountingKit 899650 1000 tests

899651 3000 tests899654 10000 tests

Alsoavailable:MTTCellProliferationAssayKit 45547A 1000 testsMTTUltraPure FP-65939A 1 g

XTTCellProliferationAssayKit FX873A 1000 testsXTTUltraPure FP-409036A 1 g

WST-1CellProliferationAssayKit KS0790 96testsWST-1asstandaloneproduct F98883 100 mg

Cellproliferationassay:

Technical tipFormazanbasedCellViabilityAssayKit

MTTbasedassayisprobablythemostpopularcell viability assay. It has several drawbacksincluding toxicity, poor solubility that requiresan extraction step and limited sensitivity. Interchim provides those kits as well (seerelatedproducts)butrecommendsstronglytheWST-8assaykit,oralternatively theUptiBluereagent.

Viability &

Cytotoxicity A

Live cell staining

■aCella™-TOXBioluminescenceCytotoxicityAssay(GAPDH)MeasurementofCell-Mediated(TCells,ADCC,NK)orComplement-MediatedCytolysis

» Versatile :Assaycanberuninserumsupplementedmedia.» Homogenous-One-step,nowashassay.Assaycanberuninsameplateassamples.» FAST-Resultsin3-5minutes.» HighlySensitive-Candetectfewerthan500cells/well.» WorkswithPRIMARYCELLSfordeterminingcellCytotoxicity.» Non-destructiveassayallowsmonitoringofadditionalparameters.

aCella-TOXprovidesanewandhighlysensitiveassayusinga patented coupled luminescent technology for the detection of cytotoxicity(1). This assay quantitatively measures the release of Glyceraldehyde-3-Phosphate Dehydrogenase(GAPDH)fromPrimaryCells,mammaliancelllines,bacterialcells(1,2,3).aCella-TOXcanworkindifferentmediaformulationsand allows overnight assays while retaining its sensitivity.The sensitivity of aCella-TOX is also greatly enhancedby the coupled luminescent signal-amplification system(3-Phosphoglyceric Phosphokinase/ATP/Luciferase), whichyields a strong luminescent signal from even small amounts of released enzyme.

In the aCella-TOX reaction scheme the release ofGAPDHis coupled to the activity of the enzyme 3-Phosphoglyceric Phosphokinase (PGK) to produce ATP. ATP is detectedvia the luciferase, luciferin Bioluminescence methodology. Further,aCella-TOXisahomogeneouscytotoxicityassay;alternativelyindualmode,aCella-TOXcanmeasurecytotoxicityandcellviabilityinthesameplate.Culturesupernatantscanalsoberemovedfromtheoriginalplateandassayedinadifferentplate,allowingkineticsrunstobesetup.Theassayisnon-destructive,allowingthemonitoringofadditionalparameterssuchasgeneexpression.Themethodishighlygeneral,sinceallknowncellsexpresscopiousamountsofGAPDH,and,unlikeotherenzymes,GAPDHisveryreadilyreleasedfromthecytoplasmuponcelllysis.Usingspeciallyadaptedformulations,thesensitivityofthemethodcanbedrivenbelow1eukaryoticcell(2).

Applications :TheaCella-TOXmethodhasbeentestedwithmanymodesofcytolysis,including:» Cellularcytotoxicity(Tcells)» Complement(2,3), pore-forming agents» Antibiotic-mediatedlysisofbacteria» Detergentmediatedandmechanicallysis

References:1.Methodsandcompositionsforcoupledluminescentassays.UnitedStatesPatent6,811,990CoreyandKinders,issuedNovember2,2004.2.Corey,M.J.andKinders,R.J.(2005),DrugDiscoveryHandbook,Ed.ShayneCoxGad,pp.689-7313.Corey,M.J.,etalJournalofImmunologicalMethods207:43-51,1997.4.Corey,M.J.,etal.,JournalofBiologicalChemistry275:12917-12925,2000.5.OgbomoH.,etal.-BiochemicalandBiophysicalResearchComunications339(2006)pp375-379.6.Corey,J.andKinders,J.(2005),DrugDiscoveryHandbook,Ed.ShayneCoxGad,pp.689-731

Jurkatcellswereplatedatvariouscellconcentrationsperwell.NP-40cytotoxicagentwasaddedtoeachwell.TheaCella-TOXkitwasusedtodetectedG3PDHenzymerelease.DatapointsshowaverageRLUintriplicate.

Description P/N : QtyaCella-ToxbioluminescentCytotoxicityAssay CA4670 500testsKitContent:4xEnzymeAssayReagent,1xEnzymeAssayDiluent,Glyeraldehyde3-Phosphate(G3P),50xDetectionReagent,5.5xDetectionAssayDiluent,LyticAgent

ATP, ADP, Phosphate & Pyrophosphate Assays

■ATPAssaykit,0.1to100pmolTodetectATPinbiologicalsamplesormonitoreATPdependentenzymeassays

Substrate:luciferinwithstabilizerλem. : 560nmSensitivity:0.1to100pmolATP - 30 min. signal

TheATPDeterminationKit, sensitiveassay,offersaconvenientbioluminescenseassay forquantitativedeterminationof smallamountsofATP.CatalysedbyfireflyluciferasethesubstrateD-luciferinisoxidizedinanATP-dependentprocessgeneratingchemiluminescenceat560nm(pH7.8):

luciferin+ATP+O2 Mg2+, luciferase Oxyluciferin+ATP+pyrophospate+CO2+light

The sensitive assay is optimized for fast determination of low levels of pre-existingATPorATP formed in kinetic systems.After a 10min incubation of the assay reagent,ATPconcentrationsdown to0.1pmolcanbeexactlydeterminedusing the linear luminescentsignaloftheluciferasereaction.Lossofluminescentsignalandsensitivityisobservedafterincubationtimesofmorethan30minutes.Ifyouareinterestedinatime-stableassay(i.e.forhighthroughputscreenings)withnearlyconstantluminescencesignalsoveraperiodofuptofourhours,useourSteadyGlowATPAssayKit.

Description P/N : QtyATPAssayKit,0.1to100pmolsensitive FP-S2841A 200-1000assays(10 ml)Eachkitcontains: FP-S2841B 600-3000assays(30 ml)ComponentA:FireflyLuciferase(readytouseglycerolstocksolution) FP-S2841C 2000-10000assays(100 ml)ComponentB:D-Luciferin(todissolveinreactionbuffer)ComponentC:DithiothreitolDTT(todissolveinreactionbuffer)ComponentD:ReactionBuffer(readytousebuffer)

Relatedproducts:ATPdisodiumsalt 00064A 25gReactionBufferasstandaloneproduct(ComponentDofATPAssayKit) CA3920 30 mlReactionBufferasstandaloneproduct(ComponentDofATPAssayKit) CA3921 100 mlARL-67156Ecto-ATPaseinhibitor CG2331 10 mg

Linear luminescence signal for ATP concentrationsdown to 0.1 pmol using theATP Determination Kit,sensitive assay.

■ATPAssayKit,SteadyGlowSubstrate:luciferinwithstabilizerλem. : 560nmSensitivity:10cells/well-10uMto0.1nMATP-4hsignal

Adenosine triphosphate (ATP) plays a fundamental role incellularenergenics,metabolicregulationandcellularsignaling.TheATPAssayKitprovidesa fast,simpleandhomogeneousluminescence assay for the determination of cell proliferation and cytotoxicity in mammalian cells. The assay can beperformedinaconvenient96-welland384-wellmicrotiter-plateformat. The high sensitivity of this assay permits the detection ofATPinmanybiologicalsystems,environmentalsamplesandfoods.ThisATPAssayKithasthestableluminescencesignalaslongas4hours.Ithasstableluminescencewithnomixingorseparationsrequired,andformulatedtohaveminimalhands-ontime.LinearluminescencesignalforATPconcentrationsfrom10µMto0.1nMwasdetectedupto5h(Z’factor=0.7)withoutsignaldecayed(abovefigshows20min,1,2,3,4,and5hrsignal).Theintegratedtimewas1sec.Description P/N : QtyATPAssayKit,SteadyGlow FN0630 96assays

Viability &

Cytotoxicity A

■ATPAssayKit,BrightGlowSubstrate:luciferinwithstabilizerλem. : 560nmSensitivity:10cells/well-3pmolATP2hincubationtime

Adenosine triphosphate (ATP) plays a fundamental rolein cellular energenics, metabolic regulation and cellularsignaling.TheATPAssayKit providesa fast, simpleandhomogeneous luminescence assay for the determination of cell proliferation and cytotoxicity in mammalian cells. The highsensitivityof thisassaypermits thedetectionofATPin many biological systems, environmental samples andfoods.ThisATPAssayKitcandetectaslowas10cells/well.It hasstable luminescencewithnomixingorseparationsrequired, and formulated to have minimal hands-on time.

ATPdoseresponseon96-wellwhiteplateusing2hincubationtime(Z’factor=0.6,Blue30min,red1h,andgreen2h).Theintegrationtimewas1sec.Thehalflifeismorethan1.5h.

Description P/N : QtyATPAssayKit,BrightGlow FN0640 96assays

■UniversalFluorimetricKinaseAssayKit,RedFluorescenceMostofcommercialproteinkinaseassaykitsareeitherbasedonmonitoringofphosphopeptideformationorATPdeletion.ForthekinaseassaykitsthatarebasedondetectionofphosphopeptidesonehastospendtimeandeffortstoidentifyanoptimizedpeptidesubstratewhiletheATPdepletionmethodsuffersvarious interferencesdue to theuseof luciferase thatare inhibitedoractivatedbyvariousbiologicalcompounds.TheUniversalKinaseAssayKitisbasedonthemonitoringofADPformation,whichisdirectlyproportionaltoenzymephoshphotransferaseactivityandismeasuredfluorimetricaly.Thiskitprovidesa fast,simple,andhomogeneousassay formeasurekinasesactivities.Thecharacteristicsof itshighsensitivity (<0.2uMADP),broadATPtolerance(1-300uM),non-antibodybased,non-radioactiveandno-washmethodtodetecttheamountofADPproducedasaresultofenzymeactivitymakeitanidealkitfordeterminingkinaseMichaelis-Mentenkineticsandforscreeningandidentifyingkinaseinhibitors.

» Universal:CanbeusedforanykinasesthatusedATPasphosphatedonor.» Continuous:Easilyadaptedtoautomationwithnomixingorseparationprotocols.» UseofNativesubstrates:Substratescanbeproteins,peptidesorsugars.» Non-Antibody-Based:Noantibodyisusedinthekit.

Description P/N : QtyUniversalFluorimetricKinaseAssayKit(540/590nm) CL9170 250assaysContains:ADPsensorbuffer,ADPSensor,ADPstandard,ADPAssayBuffer

■ADPAssayKit,RedFluorescenceSubstrate:redfluorescentsubstrateλex./em. : 571/585nmSensitivity:0.2μMADPLargeRangeofATPTolerance:1-300μM.

ADPis involvedinmanybiologicalreactionssuchasproteinkinases.OurADPassaykitcanbeusedfor assaying protein kinase reactions universallyby monitoring ADP formation, which is directlyproportional to enzyme phosphotransferase activity and is measured fluorimetrically. This kitprovides a fast, simple and homogeneous assay for measuring ADP formation or depletion. Theassayiscontinuous,andcanbeeasilyadaptedtoautomation.Thekitisconvenient,requiringminimalhands-on time. Protein kinases are of interest toresearchers involved in drug discovery due to their broad relevance to diseases such as cancer andotherproliferativediseases,inflammatorydiseases,metabolic disorders and neurological diseases.Mostof commercialproteinkinaseassaykitsareeitherbasedonmonitoringofphosphopeptideformationorATPdeletion.OurADPAssayKitisbasedonthemonitoringofADPformation,whichisdirectlyproportionaltoenzymephoshphotransferaseactivityandismeasuredfluorimetricaly.Thiskitprovidesafast,simple,andhomogeneousassayformeasurekinasesactivities.

ADPdoseresponseon384-wellblackplatewith15,30minutesand1hourincubationtime(Z’factor=0.65).

Protein kinaseAdetectionwith incubation of the kinase in thepresenceofATPandkemptidepeptidesubstratefor30minutes.

Description P/N : QtyADPAssayKit,RedFluorescence CI4171 100 assays

Viability &

Cytotoxicity A

■PhosphateAssayKit,BlueFluorescenceSubstrate:proprietarysubstrateλex./em : 370/420nm

Phosphateisinvolvedinmanybiologicalreactions.Forexample,phosphatases,ATPasesandseveralotherenzymescatalyzereactionsinwhichinorganicphosphate(Pi)isreleasedfromasubstrate.ThisPhosphateAssayKithasbeendevelopedformeasuringtheactivityofanyPi-generatingenzyme.The kit is formulated to give the simplest detection of Pi, neither coupling enzymes nor hazardous radioactivemethods are involved.ThemeasurementofPi isbasedonourproprietaryfluorescentsensorthathasitsfluorescenceintensityproportionallydependentonphosphateconcentration.Unlikeotherphosphateassays,thiskitiseasytouse.Itisamixandreadformat,andcompatiblewithallthebiologicalbuffers.

Description P/N : QtyPhosphateAssayKit,BlueFluorescence JQ8120 1kit

■PhosphateAssayKit,RedFluorescenceSubstrate:proprietarysubstrateλex./em. : 540/590nmSensitivity:0.1μMphosphate

Cellsutilizeawidevarietyofphosphate(Pi)andpolyphosphate esters as enzyme substrates,second messengers, membrane structuralcomponents and vital energy reservoirs. Phosphate is involved in many biologicalprocesses. For example, phosphatases, ATPasesandseveralotherenzymescatalyzebiochemical reactions in which inorganicphosphate is released from a phosphoester substrate. Detection of many phosphoester–metabolizing enzymes is difficult becausesuitablesubstratesarenotavailable.Itusuallyhas been necessary to determine inorganicphosphate release using tedious colorimetric assays or radioisotope-based methods. ThisFluorimetric Phosphate Assay Kit has beendeveloped for measuring the activity of any Pi-generatingenzymeusingour redfluorescentphosphate sensor. The measurement of Pi isbasedon thechange in theabsorbanceandfluorescenceofournewphosphatesensor.Ourkitprovidesall theessential reagents includingphosphatesensor,phosphatestandardsandassaybuffer.Itcanbeusedtomeasurethekineticsofphosphatereleasefromphosphatases(suchasGTPasesandATPases)bycouplingthetwoenzymaticreactions.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatandeasilyadaptedtoautomationwithnoseparationstepsrequired.

Phosphatedoseresponseon96-wellblackplatewith1hrincubationtime

Description P/N : QtyPhosphateAssayKit,RedFluorescence CI4161 100 assays

Alsoavailable:colorimetricphosphateassays*PhosphateAssay,MGmethod IS2790 1kit(600assays)Originalmolybdateandmalachytegreendyemethod.600-660nmreading.

PhosphateAssay,MGPlusmethod CI4211 1kit(1000assays)Improvedend-pointstablesignal(notpronetoprecipitation)

*The kit can also be used to estimate thephosphate content of proteins (phosphoserine or phosphothreoninepost-translationalmodifications,afteralkalinehydrolysis.

■PyrophosphateAssayKit,BlueFluorescenceSubstrate:proprietarysubstrateλex./em. : 370/420nmSensitivity:0.3μM (30 pmoles) pyrophosphate

Pyrophosphate (PPi) are produced by a numberofbiochemicalreactions,suchasATPhydrolysis,DNA and RNA polymerizations, cyclic AMPformation by the enzyme adenylate cyclase andthe enzymatic activation of fatty acids to form their coenzyme A esters. The Pyrophosphate AssayKit provides the most robust spectrophotometricmethod for measuring pyrophosphate. This kituses our proprietary fluorogenic pyrophosphatesensor that has its fluorescence intensityproportionally dependent upon the concentration of pyrophosphate. Our assay is much easierand more robust than the enzyme-couplingpyrophosphatemethods that requireat least twoenzymes for their pyrophosphate detections. Thekitprovidesall theessentialcomponents forassaying pyrophosphate.

Description P/N : QtyPyrophosphateAssayKit,BlueFluorescence JQ8080 200assays

Viability &

Cytotoxicity A

NAD/NADH, NADP/NADPH Assay Kits

Nicotinamideadeninedinucleotide(NAD+)andnicotinamideadeninedinucleotidephosphate(NADP+)aretwoimportantcofactorsfoundincells.NADHisthereducedformofNAD+,andNAD+istheoxidizedformofNADH.ItformsNADPwiththeadditionofaphosphategrouptothe2'positionoftheadenylnucleotidethroughanesterlinkage.NADPisusedinanabolicbiologicalreactions,suchasfattyacidandnucleicacidsynthesis,whichrequireNADPHasareducingagent.Inchloroplasts,NADPisanoxidizingagentimportantinthepreliminaryreactionsofphotosynthesis.TheNADPHproducedbyphotosynthesis is thenusedas reducingpower for thebiosynthetic reactions in theCalvincycleofphotosynthesis.The traditionalNAD/NADHandNADP/NADPHassaysaredonebymonitoringofNADHorNADPHabsorptionat340nm.ThismethodsufferslowsensitivityandhighinterferencesincetheassayisdoneintheUVrangethatrequiresexpensivequartzmicroplate.ThesesNAD/NADH&NADP/NADPHAssayKitsprovideaconvenientmethod forsensitivedetection. Inaddition, thisassayhasvery lowbackgroundsince it is run in theredvisible rangethatsignificantlyreducestheinterferencefrombiologicalsamples.Theassayhasdemonstratedhighsensitivityandlowinterferencewith570nmexcitation590nmemission.

■NADHAssayKit,RedFluorescenceSensitivity:10nanomolesofNADHinsolution

The enzymes in the system specificallyrecognize NADH in an enzyme cyclingreactionwhichsignificantlyincreasesdetectionsensitivity.

NADPH dose response on 96-well black plate wasmeasuredwith1hourincubationtime(n=3)whilethereisnoresponsefromNADP(theinsertshowsthelowerdetection limit).

Description P/N : QtyFluorimetricNADPHAssayKit JQ7320 400 assays

■NAD/NADHAssayKit,RedfluorescenceSensitivity:100nM(10pmol/well)ofNADHin solutionλexc./em.: 570/590nm

The enzymes in the system specificallyrecognize NAD/NADH in an enzyme cyclingreaction.ThereisnoneedtopurifyNAD/NADHfrom sample mix. The enzyme cycling reaction significantlyincreasesdetectionsensitivity.

Description P/N : QtyNAD/NADHAssayKit,Redfluorescence JQ7280 400 assays

NADH dose response on 96-well black plate wasmeasuredwith1hourincubationtime(n=3)whilethereisnoresponsefromNADPH.

NAD/NADH, NADP/NADPH Assay Kits

■NADPHAssayKit,RedFluorescenceSensitivity:30nM(0,3nmol/well)ofNADPHinsolution

The enzymes in the system specificallyrecognize NADPH in an enzyme cyclingreaction. The enzyme cycling reaction significantlyincreasesdetectionsensitivity.

NADPHdoseresponseon96-wellblackplatewasmeasuredwith 1 hour incubation time (n=3)whilethereisnoresponsefromNADP(theinsertshowsthelowerdetectionlimit).

Description P/N : QtyFluorimetricNADPHAssayKit,Redfluorescence JQ7330 400 assays

■NADP/NADPHAssayKit,RedfluorescenceSensitivity:10nM(1pmol/well)ofNADPHin solution

This NADP/NADPH Assay Kit provides aconvenient method for sensitive detection of NADP,NADPHandtheirratio.Theenzymesin the system specifically recognize NADP/NADPHinanenzymecyclingreaction.Thereis no need to purify NADP/NADPH fromsample mix. The enzyme cycling reaction significantlyincreasesdetectionsensitivity.

NADPH dose response on 96-well black plate wasmeasuredwithNADP/NADPHAssayKitwith 30minincubationtime(n=3)whilethereisnoresponsefromNADH.

Description P/N : QtyNADP/NADPHAssayKit,Redfluorescence JQ7300 400 assays

cell counting, viability & proliferation

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