Viability & Cytotoxicity Assays 28 votre source d’innovations .com .com Cell counting, viability & proliferation Technical tip Cell counting is required » To monitor cells during cell cultures » For cell preparation or any cell experiment » To standardize cell samples for analysis. » Cell proliferation » Cytotoxicity assays Several methods have been proposed, each fitting more or less to each specific application : counting dead cells may be acceptable for the preparation of cell extracts or desired when one do not want to operate with hazardous cells or for cytotoxicity study. At the opposite dead cells counting is generally precluded for cell culture and bioassays. It may be useful to quantitate only viable cells, or only fast proliferating cells. Interchim provides a large choice of cell assays covering standard as well as innovative methods for general to specific cell assays. Selection guide Probe Principle Detection Method Dead Viable Proliferating Features/Advantages - Drawbacks Trypan blue Membrane exclusion Colorimetric ++ ++ ++ Cheap, but time consuming, not scalable. Microscopy Do not state on viability. Hoechst DNA probe exclusion Fluorimetric ++ ++ +++ Cheap, Scalable, Non toxic. Do not state on viability. More rapid than MTT/XTT ; unfixed or fixed samples. MTT Formazan dye, orange precipitate. Colorimetric - ++ +++ Popular method. Sensitive, Scalable. Non toxic Increased solubility and performance from MTT to XTT and WST. XTT Same as MTT but more soluble. Colorimetric - ++ +++ WST Formazan dye, soluble & not toxic - ++ +++ UptiBlue ratiometric blue probe Colorimetric - +++ +++ No solubilization step (unlike MTT). Applyalso to for cell redox Fluorimetric adherent cells. Sensitivity similar to MTT/XTT, but easier to use Fluorimetry/Superior sensitivity to MTT / XTT. Calcein-AM Calcein accumulation in Fluorimetric - +++ ++ No solubilization step (unlike MTT/XTT). Adaptable to a cytoplasm wide variety of techniques, including : microplate assays, in vivo cell tracing. Do not work for bacteria. May alter some cell functions. GAPDH Release of GAPDH coupled to ATP assay Bioluminescence - +++ + Measurement of Cell-Mediated (T Cells, ADCC, NK) or Complement-Mediated Cytolsis. CFSE Fluorescein protein labeling Fluorimetric ++ ++ ++ Useful when other method do not work properly. Do not state on viability. AnnexinV AnnexinV/PhosphoSerine Fluorimetric + +++ + Useful for Apoptosis study. LDH convertion in colored product - ++ + Recommended for cytotoxicity assays Serum Interference. Luciferin Syst. ATP measure Luminescence - + +++ Pros : sensitivity / linearity. Cons : signal depends on each cell line, on temperature -3H Thymidine DNA incorporation of Radioactivity - + +++ Cons : hazardous (radioelements). radioactivity BRDU DNA incorporation Immunoassay - + +++ 51 Cr release EU 3+ Release of radioactivity by Radioactivity - - +++ Recommended for cytotoxicity assays. cytoplasm Cons : hazardous (radioelements). Propidium 7-Membrane permeability Fluorimetric +++ - - Used in combinaison of green fluorescence dye like. Iodide, AAD Annexin V-FP488 to discriminate dead cells from alive cells. MicroPlate readers & Imaging systems Interchim and Berthold collaboration supports further your works. Many of our fluorescence and luminescence reagents and kits were validated with instruments. *NightOWL LB983 NC100 *Mithras LB940 MultiMode Reader
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Via
bilit
y &
Cyt
otox
icity
Ass
ays
28 votre source d’innovations.com .co
m votre source d’innovations
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.
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.
» 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.
■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: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).
Via
bilit
y &
Cyt
otox
icity
Ass
ays
32 votre source d’innovations.com .co
m votre source d’innovations
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
■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.
■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
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.
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).
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.
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
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.
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.
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.
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.
*The kit can also be used to estimate thephosphate content of proteins (phosphoserine or phosphothreoninepost-translationalmodifications,afteralkalinehydrolysis.
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.
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.
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).
The enzymes in the system specificallyrecognize NAD/NADH in an enzyme cyclingreaction.ThereisnoneedtopurifyNAD/NADHfrom sample mix. The enzyme cycling reaction significantlyincreasesdetectionsensitivity.
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).
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.