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Ex/Em(nm)	646/659
MW	~500
CAS#	N/A
Solvent	DMSO
Storage	F/D/L
Category	CellBIOLOGy LabelingCells
Related	ViABIlityandProliferation CellViability MDRResearch BiochemicalAssays

CalceinAMisonethemostpopularfluorescentprobesusedforlabelingandmonitoringcellularfunctionsoflivecells.However,thesinglecolorofCalceinAMmakesitimpossIBLetousethisvaluablereagentinthemulticolorapplications.Forexample,itisimpossibletouseCalceinAMincombinationofGFP-tranfactedcellsduetothesamecolortoGFP.ToaddressthiscolorlimitationofCalceinAM,wehavedevelopedCalceinOrange™,CalceinRed™andCalceinDeepRed™.ThesenewCalceinAManalogsenablethemulticolorlabelingandfunctionalanalysisoflivecellsincombinationwithCalceinAM.Non-fluorescentCalceinDeepRed™acetatecaneasilygetintolivecellsandhydrolyzestogeneratestronglyfluorescentCalceinDeepRed™(Cat#:21902)dye.CalceinDeepRed™dyecanbemonitoredwiththecommonCy5filterset.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparecells:

Plate100to10,000cellsperwellina96-wellblackwall/clearbottommicroplate.Addtestcompoundsintothecellsforadesiredperiodoftime(suchas24,48or96hours)ina37°C,5%CO₂incubator.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.Thetotalsuggestedvolumeis100µLfora96-wellplate,and25µLfora384-wellplate.

Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforproliferationorcytotoxicityinduction.Forproliferation,usefewercells,andfortoxicityassays,usemorecellstostartwith.

 

2.PrepareCalceinDeepRed^™workingsolution(for1plate):

2.1   Preparea2to5mMstocksolutionofCalceinDeepRed^™inhigh-quality,anhydrousDMSO.Thestocksolutionshouldbeusedpromptly;anyremainingsolutionshouldbealiquotedandrefrozenat<-20^oC.

Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.

 

2.2   PrepareCalceinDeepRed^™workingsolution:Onthedayoftheexperiment,eitherdissolveCalceinDeepRed^™solidinDMSOorthawanaliquotoftheCalceinDeepRed^™stocksolutiontoroomtemperature.Preparea5to10µMCalceinDeepRed^™workingsolutioninHanksand20mMHepesbuffer(HHBS)orbufferofyourchoicepH7,with2.5mMprobenicid.

Note1:10mLofCalceinDeepRed^™workingsolutionisnotstableatroomtemperature;preparefreshbeforeuse.

Note2:Manycellscontainorganic-aniontransporters.Itishighlyrecommendedtoaddadditional2.5mMprobenecid,aninhibitoroforganic-aniontransporters,intheCalceinDeepRed^™workingsolution.

 

3.Runthecellviabilityassay:

3.1   Treatcellswithtestcompoundsasdesired(fromStep1).

Note:Itisnotnecessarytowashcellsbeforecompoundaddition.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds,providedresidualvolumesaftertheaspiratestep.Alternatively,cellscanbegrowninserum-freemedia.

 

3.2    Removethemediumfromthecells.

Note:MediummustberemovedbeforedyeloADIng.

 

3.3   Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofCalceinDeepRed^™workingsolution(fromStep2.2).

 

3.4   IncubatetheCalceinDeepRed^™dye-loadingplateatroomtemperatureor37^oCfor1hr,protectedfromlight.

Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.

Note2:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffaftertheincubationofthedye.

 

3.5   MonitorthefluorescencechangeatEx/Em=635/670nm.

Note:Ifthebackgroundishigh,replacetheCalceinDeepRed^™workingsolution(fromStep3.4)withHHBScontaining2.5mMprobenicidbeforemonitoringthefluorescencesignal.

References&Citations

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Functionalevidencethattheself-renewalgeneNANOGregulatesesophagealsquamouscancerdevelopment
Authors:DengLi,XiaocongXiang,FeiYang,DongqinXiao,KangLiu,ZhuChen,RuolanZhang,GangFeng
Journal:BiochemicalandBiophysicalResearchCommunications(2017)

NINJ2--Anovelregulatorofendothelialinflammationandactivation
Authors:JingjingWang,JingjingFa,PengyunWang,XinzhenJia,HuixinPeng,JingChen,YifanWang,ChenhuiWang,QiuyunChen,XinTu
Journal:CellularSignalling(2017)

ErythropoietinStimulatesEndothelialProgenitorCellstoInduceEndothelializationinanAneurysmNeckAfterCoilEmbolizationbyModulatingVascularEndothelialGrowthFactor
Authors:PeixiLiu,YingjieZhou,QingzhuAn,YayingSong,XiChen,Guo-YuanYang,WeiZhu
Journal:MEDICINE(2016):1--8

FlexibleEndoscopicSprayApplicationofRespiratoryEpithelialCellsasPlatformTechnologytoApplyCellsinTubularOrgans
Authors:AnjaLenaThiebes,ManuelArminReddemann,JohannesPalmer,ReinholdKneer,StefanJockenhoevel,ChristianGabrielCornelissen
Journal:TissueEngineeringPartC:Methods(2016):322--331

Influenceofhypothermiaandsubsequentrewarminguponleukocyte-endothelialinteractionsandexpressionofJunctional-Adhesion-MoleculesAandB
Authors:NicolaiVBogert,IsabellaWerner,AngelaKornberger,PatrickMeybohm,AntonMoritz,TillKeller,UlrichAStock,AndresBeiras-Fernandez
Journal:Scientificreports(2016)

Influenceofhypothermiaandsubsequentrewarminguponleukocyte-endothelialinteractionsandexpressionofJunctional-Adhesion-MoleculesAandB
Authors:NicolaiVBogert,IsabellaWerner,AngelaKornberger,PatrickMeybohm,AntonMoritz,TillKeller,UlrichAStock,AndresBeiras-Fernandez
Journal:Scientificreports(2016)

InhibitionofABCtransportproteinsbyoilsandsprocessaffectedwater
Authors:HattanAAlharbi,DavidMVSaunders,AhmedAl-Mousa,JaneAlcorn,AlbertoSPereira,JonathanWMartin,JohnPGiesy,SteveBWiseman
Journal:AquaticToxicology(2016):81--88

Rapidgenerationofcollagen-basedmicrotissuestostudycell--matrixinteractions
Authors:Marie-ElenaBrett,AlexandraLCrampton,DavidKWood
Journal:Technology(2016):1--8

ToxicokineticsandtoxicodynamicsofchlorpyrifosisalteredinembryosofJapanesemedakaexposedtooilsandsprocess-affectedwater:evidenceforinhibitionofP-glycoprotein
Authors:HattanAAlharbi,JaneAlcorn,AhmedAl-Mousa,JohnPGiesy,SteveBWiseman
Journal:JournalofAppliedToxicology(2016)

Sprayingrespiratoryepithelialcellstocoattissue-engineeredconstructs
Authors:AnjaLenaThiebes,StefanieAlbers,ChristianKlopsch,StefanJockenhoevel,ChristianGCornelissen
Journal:BioResearchopenaccess(2015):278--287

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