Overview | ![]() PrinterFriendlyVersion |
Ex/Em(nm) | 646/659 |
MW | ~350 |
CAS# | N/A |
Solvent | DMSO |
Storage | F/D/L |
Category | CellBIOLOGy CellFunctionalAnalysis |
Related | HydrolyticEnzymes BiochemicalAssays |
Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
Plate100to100,000x10cellsperwellinatissueculturemicroplatewithblackwallandclearbottom.Addtestcompoundsintothecellsandincubateforadesiredperiodoftime(suchas24,48or96hours)ina37°C,5%CO2incubator.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.Thesuggestedtotalvolumeis100µL/well/96-wellplate,and25µL/well/384-wellplate.
Note:Eachcelllineshouldbeevaluatedontheindividualbasistodeterminetheoptimalcelldensityforproliferationorcytotoxicityinduction.Forproliferationassays,usefewercells;forcytotoxicityassays,usemorecellstostartwith.
2.PrepareSunRed™acetateworkingsolution:
2.1 Preparea2to10mMstocksolutionofSunRed™acetateinDMSO.Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandfrozenat<-20oC.Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
2.2 PrepareSunRed™acetateworkingsolutionat5to10µMin1XHank"ssaltsolutionwith20mMHepesbuffer(HHBS)orbufferofyourchoicebeforetheexperiment.
3.Runthecellviabilityassay:
3.1 Treatcellswithtestcompoundsasdesired(fromStep1).
Note:Itisnotnecessarytowashcellsbeforeaddingcompound.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds.Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofHHBSorthebufferofyourchoiceafteraspiration.Alternatively,cellscanbegrowninserum-freemedia.
3.2 Removethemediumfromthecells.
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofSunRed™acetateworkingsolution(fromStep2.2).
3.4 IncubatetheSunRed™acetateworkingsolutionplateatroomtemperatureor37oCfor1hour,protectedfromlight.
Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
Note2:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffafterincubation.
3.5 MonitorthefluorescenceintensityatEx/Em=620/660nm(cutoff640nm)
References&Citations | ![]() PrinterFriendlyVersion |
1. ZhuH,LiuM,SumbyP,LeiB.(2009)Thesecretedesteraseofgroupastreptococcusisimportantforinvasiveskininfectionanddisseminationinmice.InfectImmun,77,5225.
2. ReadDJ,LiY,ChaoMV,CavanaghJB,GlynnP.(2009)Neuropathytargetesteraseisrequiredforadultvertebrateaxonmaintenance.JNeurosci,29,11594.
3. MoronoY,TakanoS,MiyanagaK,TanjiY,UnnoH,HoriK.(2004)Applicationofglutaraldehydeforthestainingofesterase-activecellswithcarboxyfluoresceindiacetate.BiotechnolLett,26,379.
4. LourencoMF,CeronCR,CararetoCM.(2001)EvaluationoffitnesscomponentsinstrainsofDrosophilamullericarryingdifferentgenotypesforanesterase.Cytobios,106,125.
5. StewardN,MartinR,EngasserJM,GoergenJL.(1999)DeterminationofgrowthandlysiskineticsinplantcellsUSPensionculturesfromthemeasurementofesteraserelease.BiotechnolBioeng,66,114.
6. DegrassiG,UotilaL,KlimaR,VenturiV.(1999)PurificationandpropertiesofanesterasefromtheyeastSaccharomycescerevisiaeandidentificationoftheencodinggene.ApplEnvironMicrobiol,65,3470.
7. BarberD,CorrellL,EhrichM.(1999)Comparisonoftwoinvitroactivationsystemsforprotoxicantorganophosphorousesteraseinhibitors.ToxicolSci,47,16.
8. LiW,CasidaJE.(1998)Organophosphorusneuropathytargetesteraseinhibitorsselectivelyblockoutgrowthofneurite-likeandcellprocessesinculturedcells.ToxicolLett,98,139.
9. OakeshottJG,SaadM,GameAY,HealyMJ.(1994)Causesandconsequencesofesterase6enzymeactivityvariationinpre-adultDrosophilamelanogaster.Heredity,73(Pt2),160.
10. TotskiiVN,EserkepovaEV,DzhanZU.(1994)[Esterase-6gene-enzymesystemandresistanceofDrosophilatoincreasedtemperature].Genetika,30,342.
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