EZQuant™CytotoxicityAssayKit:#CA01
ProductDescription
TheEZQuant™CytotoxicityAssayKitisusedtoassesscelldeath.ItmeasuresthelevelofLDHreleasedwhencellsundergostressorinjuryfromchemicalsthataretoxictothecells.ThereagentisreducedbyNADHthatwasoxidizedbytheextracellularLDH.Therefore,theamountofformazanproducedisdirectlyproportionaltothenumberofdeadcells.
PairwithEZQuant™CellQuantifyingKit
TheEZQuant™CellQuantifyingKitconvenientlydeterminesthenumberofviablecellsincellproliferationandcytotoxicityassays.Thisproductisusefulfordrugscreeningandcytotoxicitytestingofchemicals.Meanwhile,theEZQuant™CytotoxicityAssayKitdeterminesthenumberofdamagedcellsandthedegreeofcytotoxicity.Combiningthesetwoproductswillallowresearcherstofullyunderstandthecellhealthconditionsundervariouscytotoxicenvironments.
Features- Highlinearityrange
- Longshelf-lifewithoutdegradation(2monthsat0-5°C)
- Simplehomogenousassayornon-homogenous(multiplex)assaypossIBLe
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ClicktoseeourRESULTS.
ProductSpecifications |
ProductName | CytotoxicityAssayKit |
Catalog# | CA01 |
Size | 500tests(CA01)/2000tests(CA04) |
Shipping | RoomTemperature |
StorageandStABIlity | Storeat0-5°C |
QualityControl | Appearance,blank,andsensitivitytestsareperformedforeachlot.Onlylotsthatpassthefollowingcriteriaareoffered:BlankAbsorbance(460nm)≤0.700SensitivityAbsorbance(460nm)≥1.000 |
RestrictedUse | ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures. |
Protocol | Download |
- Homogenousassay
- Non-homogenousassay
CellConcentrationOptimization- Collectcellsandwashthemwiththeassaymedium.PreparecellsUSPensionto5×105cells/mlintheassaymedium.
- Add100μloftheassaymediumtoeachwellofaflat-bottom96-welltissuecultureplate.
- Prepare2-foldserialdilutionofeachwellintriplicatesetofwellsforthehigh-control,low-controlandbackgroundcontrol(mediumonly).Serialdilution:Addthecellsuspension(5×105cells/ml)tothefirstwellandmixbypipetting.Thiswellcontainsthemaximumnumberofcells(2.5×104cells/well).Transfer100μlfromthefirstwelltothenextwell,andmixbypipetting.Repeatthisprocedure.
- Incubatetheplateat37°CforanappropriatetimeinaCO2incubator.Usethesameincubationtimeinthecytotoxicityassay.
- Add10μlofthelysisbuffertoeachwellofthehighcontrol.
- Incubatetheplateat37°Cfor30minutesinaCO2incubator.
- Add100μloftheworkingsolutiontoeachwell.Protecttheplatefromlightandincubateitattheroomtemperaturefor30minutes.
- Add50μlofthestopsolutiontoeachwell.
- Measuretheabsorbanceat490nmbyamicroplatereader.
CytotoxicityAssay- Add50μlofcellsuspensiontoeachwellofaflat-bottom96-welltissuecultureplate.Foradherentcells:incubatetheplateat37°CovernightinaCO2incubatortoallowthecellstoadhereandthenreplacetheassaymediumwith50μloffreshassaymedium.
- Add50μlofassaymediumcontainingtestsubstancethatadjustedtothedesiredconcentration.
| Backgroundcontrol | Testsubstance | Lowcontrol | Highcontrol |
Assaymedium | 100μl | N/A | 50μl | 50μl |
Cellsuspension | N/A | 50μl | 50μl | 50μl |
Testsubstanceinculturemedium | N/A | 50μl | N/A | N/A |
Lysisbuffer | N/A | N/A | N/A | 10μl |
- Incubatetheplateat37°CforanappropriatetimeperiodinaCO2incubator.
- Add10μlofthelysisbuffertoeachwellofthehighcontrol.Incubatetheplateat37°Cfor30minutesinaCO2incubator.
- Add100μloftheworkingsolutiontoeachwell.Protecttheplatefromlightandincubateitattheroomtemperaturefor30minutes.
- Add50μlofthestopsolutiontoeachwell.
- Measuretheabsorbanceat490nmbyamicroplatereader.
CalculateCytotoxicityCalculatetheaverageabsorbancefromeachtriplicatesetofwellsandsubtractthebackgroundcontrolvaluefromeachabsorbanceone.Calculatethepercentcytotoxicitybythefollowingequation:Cytotoxicity(%)=(Testsubstance-Lowcontrol)/(Highcontrol-Lowcontrol)x100
CellConcentrationOptimization- Collectcellsandwashthemwiththeassaymedium.Preparecellsuspensionto5×105cells/mlintheassaymedium.
- Add100μloftheassaymediumtoeachwellofa96-welltissuecultureplate.Useroundorv-bottomedplateforsuspensioncells,flat-bottomedplateforadherentcells.
- Prepare2-foldserialdilutionofeachwellintriplicatesetofwellsforthehigh-control,low-controlandbackgroundcontrol(mediumonly).Serialdilution:Addthecellsuspension(5×105cells/ml)tothefirstwellandmixbypipetting.Thiswellcontainsthemaximumnumberofcells(2.5×104cells/well).Transfer100μlfromthefirstwelltothenextwell,andmixbypipetting.Repeatthisprocedure.
- Add100μloftheassaymediumtoeachwell.
- Incubatetheplateat37°CforanappropriatetimeinaCO2incubator.Usethesameincubationtimeinthecytotoxicityassay.
- Add20μlofthelysisbuffertoeachwellofthehighcontrol.
- Incubatetheplateat37°Cfor30minutesinaCO2incubator.
- Centrifugetheplateat250×gfor2minutestoprecipitatethecells(forsuspensioncells).
- Transfer100μlofthesupernatantfromeachwelltoanopticallyclear96-wellplate.
- Add100μloftheworkingsolutiontoeachwell.Protecttheplatefromlightandincubateitattheroomtemperaturefor30minutes.
- Add50μlofthestopsolutiontoeachwell.
- Measuretheabsorbanceat490nmbyamicroplatereader.
CytotoxicityAssay- Add100μlofthecellsuspensiontoeachwellofa96-welltissuecultureplate.Foradherentcells:incubatetheplateat37°CovernightinaCO2incubatortoallowthecellstoadhereandthenreplacetheassaymediumwith100μloffreshassaymedium.
- Add100μlofassaymediumcontainingtestsubstancethatadjustedtothedesiredconcentration.
| Backgroundcontrol | Testsubstance | Lowcontrol | Highcontrol |
Assaymedium | 220μl | 20μl | 120μl | 100μl |
Cellsuspension | N/A | 100μl | 100μl | 100μl |
Testsubstanceinculturemedium | N/A | 100μl | N/A | N/A |
Lysisbuffer | N/A | N/A | N/A | 20μl |
- Incubatetheplateat37°CforanappropriatetimeperiodinaCO2incubator.
- Add20μlofthelysisbuffertoeachwellofthehighcontrol.Incubatetheplateat37°Cfor30minutesinaCO2incubator.
- Centrifugetheplateat250×gfor2minutestoprecipitatethecells(forsuspensioncells).
- Transfer100μlofthesupernatantfromeachwelltoeachwellofanewopticallyclear96-wellplate.
- Add100μloftheworkingsolutiontoeachwell.Protecttheplatefromlightandincubateitatroomtemperaturefor30minutes.
- Add50μlofthestopsolutiontoeachwell.
- Measuretheabsorbanceat490nmbyamicroplatereader.
CalculateCytotoxicityCalculatetheaverageabsorbancefromeachtriplicatesetofwellsandsubtractthebackgroundcontrolvaluefromeachabsorbanceone.Calculatethepercentcytotoxicitybythefollowingequation:Cytotoxicity(%)=(Testsubstance-Lowcontrol)/(Highcontrol-Lowcontrol)x100