Overview

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1.Preparecellsandtestcompounds:

1.1   Plate100to10,000cellsperwellinatissueculturemicroplatewithblackwallandclearbottom.Addtestcompoundsintothecells,andincubateforadesiredperiodoftime(suchas24,48or96hours)ina37°C,5%CO₂incubator.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.Thesuggestedtotalvolumeis100µLfora96-wellplateand50µLfora384-wellplate.

1.2   Setupthefollowingcontrolsatthesametime.

•Positivecontrolcontainscellsandknownproliferationorcytotoxicityinducer.

•Negativecontrolcontainscellsbutnotestcompounds.

•Vehiclecontrolcontainscellsandthevehicleusedtodelivertestcompounds.

•Non-cellcontrolcontainsgrowthmediumwithoutcells.

•Testcompoundcontrolcontainsthevehiclecontrolusedtodelivertestcompounds[Hank’sbalancesaltsolution(HBSS)orphosphate-bufferedsaline(PBS)]andtestcompound.Sometestcompoundshavestrongautofluorescenceandmaygivefalsepositiveresults.

Note:Matchthetotalvolumeofallthecontrolsto100µLfora96-wellplateor50µLfora384-wellplatewithgrowthmedium.

2.Assayprocedures:

2.1   ThawandwarmuptheAssaySolution(ComponentA)to37°C.Mixitthoroughlybeforestartingtheexperiments.

2.2   Add20µL(96-wellplate)or10µL(384-wellplate)ofAssaySolution(ComponentA)intoeachwell.Mixthereagentsbyshakingtheplategentlyfor30seconds.

2.3    Incubatethecellsina37^oC,5%CO₂incubatorfor1-24hours,protectedfromlight.

Note1:Theappropriateincubationtimedependsonthemetabolismrateoftheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.

Note2:Extremelyprolongedincubationtimeisnotrecommendedsincetheindicatorcouldbeconvertedtocolorlesscompound.

2.4   Monitortheabsorbancechangeat570nmand605nm.TheratioofOD₅₇₀toOD₆₀₅isusedtodeterminethecellviabilityineachwell.

Note:ThecellviabilityisproportionaltoincreasedOD₅₇₀anddecreasedOD₆₀₅.

3.Performdataanalysis:

3.1   Thebackgroundabsorbancereadingfromthenon-cellcontrolwellissubtractedfromthevaluesforthosewellscontainingthecells.

Note:Thebackgroundabsorbanceoftheblankwellsmayvarydependingonthesourcesofthegrowthmediaorthemicrotiterplates.

3.2   Theabsorbancereadingineachwellindicatesthecellnumberinthewell.

3.3   Calculatethepercentageofcellviabilityforsamplesandcontrolsbasedonthefollowingformula:

%Cellviability=100×(R_sample-R_o)/(R_ctrl-R_o)

R_sampleistheabsorbanceratioofOD₅₇₀/OD₆₀₅inthepresenceofthetestcompound.

R_ctrlistheabsorbanceratioofOD₅₇₀/OD₆₀₅intheabsenceofthetestcompound(vehiclecontrol).

R_oistheaveragedbackground(non-cellcontrol)absorbanceratioofOD₅₇₀/OD_605.

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