Cell viability assays

Viability Assays

1. CellViabilityAssays
Department of Pharmacology

2. Viabilityassays
•CellViabilityAssayisahomogeneousmethodtodeterminethe
numberofviablecellsinculture.
•Cellviability,definedasthenumberofhealthycellsinasample,
determinestheamountofcells(regardlessofphasearoundthecell
cycle)thatarelivingordead,basedonatotalcellsample.
•Cellviabilityandcytotoxicityassaysareusedfordrugscreening
andcytotoxicitytestsofchemicals.

3. Theyarebasedonvariouscellfunctionssuchas
•Enzymeactivity,
•Cellmembranepermeability,
•Celladherence,ATPproduction,
•Co-enzymeproduction
•Nucleotideuptakeactivity

4. DNAsynthesiscellproliferationassays
•Oneofthemostreliableandaccurateassaytypesismeasurement
ofDNAsynthesizedinthepresenceofalabel.
•Traditionalcellproliferationassaysinvolveincubatingcellsfora
fewhourstoovernightwith3H-thymidine.
•Proliferatingcellsincorporatetheradioactivelabelintotheir
nascentDNA,whichcanbewashed,adheredtofiltersandthen
measuredusingascintillationcounter.

5. Metaboliccellproliferationassays
•Anothermeasureofcellproliferationisthemetabolicactivityofa
populationofcells.
•TetrazoliumsaltsorAlamarBluearecompoundsthatbecome
reducedintheenvironmentofmetabolicallyactivecells,forming
aformazandyethatsubsequentlychangesthecolorofthemedia.
•Thisiscausedbyincreasedactivityoftheenzymelactate
dehydrogenaseduringproliferation.
•Theabsorptionofthemedia-containingdyesolutioncanberead
usingaspectrophotometerormicroplatereaderinlow-orhigh-
throughputconfigurations.

6. •Fourtypesoftetrazoliumsaltsaremostcommon
MTT
XTT
MTS
WST1
•AdisadvantageofMTTisthatitisinsolubleinstandardculture
medium,andtheformazancrystalsproducedduringreductionmust
bedissolvedinDMSOorisopropanol.
•Becauseofthis,MTTismainlyanendpointassay.
•Theothersalts,aswellasAlamarBlue,aresolubleinculturemedia
andarenontoxic.
•Theycanbeusedforcontinuousmonitoring,tofollowdynamic
changesinproliferationovertime.

7. •XTTreduceslessefficientlyandmayneedadditionalfactorsadded.
•WST1ismoresensitive,reducesmoreefficientlyandshowsfaster
colordevelopmentcomparedtotheothersalts.
•AlamarBlueisalsosensitive,capableofdetectingasfewas100cells
inawellofamicrotiterplate.
•ThetetrazoliumsaltsandAlamarBlueredoxdyescanbequantified
witharangeofinstrumentsforconventionalorhigh-throughput
studies
•Forexample,standardspectrophotometersorspectrofluorometersor
platereadersforspectrophotometricorspectrofluorometricmicrotiter
wellplates.

8. TetrazoliumReductionAssays
•Themostcommonlyusedcompoundsinclude:MTT,MTS,XTT,and
WST-1.
•Thesecompoundsfallintotwobasiccategories:
MTTwhichispositivelychargedandreadilypenetrates
viableeukaryoticcellsand
ThosesuchasMTS,XTT,andWST-1whicharenegatively
chargedanddonotreadilypenetratecells.
•Thelatterclass(MTS,XTT,WST-1)aretypicallyusedwithan
intermediateelectronacceptorthatcantransferelectronsfromthe
cytoplasmorplasmamembranetofacilitatethereductionofthe
tetrazoliumintothecoloredformazanproduct.

9. MTTTetrazoliumAssayConcept
•Thisisacolorimetricassaythatmeasuresthereductionofyellow3-
(4,5-dimethythiazol2-yl)-2,5-diphenyltetrazoliumbromide(MTT)
bymitochondrialsuccinatedehydrogenase.
•TheMTTentersthecellsandpassesintothemitochondriawhereit
isreducedtoaninsoluble,coloured(darkpurple)formazan
product.
•Thecellsarethensolubilisedwithanorganicsolvent(eg.
isopropanol)andthereleased,solubilisedformazanreagentis
measuredspectrophotometrically.
•SincereductionofMTTcanonlyoccurinmetabolicallyactivecells
thelevelofactivityisameasureoftheviabilityofthecells

11. MeasuringATP
•IttakesadvantageofthetightregulationofintracellularATPwithin
cells.
•DyingordeadcellscontainlittletonoATP,sothereisatightlinear
relationshipbetweencellnumberandtheconcentrationofATP
measuredinacelllysateorextract.
•Thebioluminescence-baseddetectionofATP,usingtheenzyme
luciferaseanditssubstrateluciferin,providesaverysensitivereadout.
•InthepresenceofATP,luciferaseproduceslight(proportionaltothe
ATPconcentration)thatcanbedetectedbyaluminometerorany
microplatereadercapableofreadingluminescentsignals.
•Thisapproachisalsowellsuitedtohigh-throughputcellproliferation
assaysandscreening.

12. •ATPhasbeenwidelyacceptedasavalidmarkerofviablecells.
•Whencellslosemembraneintegrity,theylosetheabilityto
synthesizeATPandendogenousATPasesrapidlydepleteany
remainingATPfromthecytoplasm.
•TheATPdetectionreagentcontainsdetergenttolysethecells,
ATPaseinhibitorstostabilizetheATPthatisreleasedfromthe
lysedcells,luciferinasasubstrate,andthestableformofluciferase
tocatalyzethereactionthatgeneratesphotonsoflight.

14. SulforhodamineBCellCytotoxicityAssay
•ThisassayreliesontheabilityofSRBtobindcellularprotein
componentsandmeasurethetotalbiomass.
•SRBisabright-pinkaminoxanthenedyethatcanformanelectrostatic
complexwithbasicaminoacidresiduesofproteinsinslightlyacidic
conditionsbutitcandissociateunderbasicconditions.
•Ithasbeenwidelyusedfordrugtoxicityscreeningagainstdifferent
typesofcancerousandnon-cancerouscelllines.
•Inaddition,thisassayisindependentofcellmetabolicactivityand
thereforeshouldshowlessinterferencebythetestingcompounds.
•SincethebindingofSRBisstoichiometric,theincorporateddye
releasedfromstainedcellsafterwashingisdirectlyproportionaltothe
cellbiomassandcanbemeasuredat565nm

17. Plasmamembraneintegrity
•Assessingcellmembraneintegrityisoneofthemostcommonand
straightforwardwaystomeasurecellviabilityandassess
cytotoxicconsequences.
•Compoundsthathavecytotoxiceffectsoftencompromisecell
membraneintegrityandinducenecrosis.
•Dyes,suchaspropidiumiodideand7-AAD,arenormally
excludedfromtheinsideofhealthycells;however,ifthecell
membranehasbeencompromised,theyfreelycrossthe
membraneandstainintracellularcomponents.
•Thismethoddistinguisheshealthycellswithuncompromised
membraneintegrity(unlabeled)fromnon-healthyones(colored).