Overview

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Ex/Em(nm)	646/659
MW	~350
CAS#	N/A
Solvent	DMSO
Storage	F/D/L
Category	CellBIOLOGy CellFunctionalAnalysis
Related	HydrolyticEnzymes BiochemicalAssays

Esterase-catalyzedhydrolysisofSunRedacetate(SRA)yieldstheSunRedfluorophorethatcanbeexcitedwiththe633nmlaserwithemissionof~660nm.ThefluorescenceofSunRedcanbereADIlydetectedusingtheCy5filtersetthatiscommonlyequippedwithmostofthecommercialfluorescenceinstruments.AlthoughSunRedisreadilyexcitedat633nmwithredemissionof~660nm,SRAhasveryminimalabsorptionat633nmwithoutredemission,makingSRAoneofthemostsensitiveNIResterasesubstrates.SRAprovidesasecondcolorforcellviABIlityassaywhilethepopularfluoresceincolorcanusedforanothercellularfunctionalassay.SRAisanon-fluorescenthydrophobiccompoundthatcanpassthroughthecellmembranewhereuponintracellularesteraseshydrolyzetheacetategroupproducingthehighlyfluorescentproductSunRed.TheSunRedmoleculeaccumulatesincellsthatpossessintactmembranessothedeepredfluorescencecanbeusedasaMarkerofcellviability.Cellsthatdonotpossessanintactcellmembraneoranactivemetabolismmaynotaccumulatethefluorescentproductandthereforedonotexhibitdeepredfluorescence.SRAmaybeusedincombinationwithagreenvitalstainsuchasaFITCorAlexaFluor488-labeledantibody.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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<-20^oC.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™acetateworkingsolutionplateatroomtemperatureor37^oCfor1hour,protectedfromlight.

Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.

Note2:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffafterincubation.

 

3.5   MonitorthefluorescenceintensityatEx/Em=620/660nm(cutoff640nm)

References&Citations

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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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