Description

Therearefivefluorescentcontrolliposomeproducts(Fluoroliposome®)forClodrosome®(Clodronateliposomes).Allfivefluorescentliposomesincorporatealipophilicdyeinsidetheirmembranes.Theyareinsolubleinwater;however,theirfluorescenceiseasilydetectedwhenincorporatedintomembranes.DiI,DiO,DiD,DiRandDiAcoverawiderangeofexcitationandemissionwavelengthsfrom300sto900s.DiIandDiOhavefluorescenceexcitationandemissionmaximaseparatedbyabout65nm,facilitatingtwo-colorlabeling.TheemissionspectrumofDiAisverybroad,allowingittobedetectedasgreen,orangeorevenredfluorescencedependingontheopticalfilterused.DiI,DiO,DiDandDiRbelongtothedialkylcarbocyaninesfamilyofcompounds.Thespectralpropertiesofthedialkylcarbocyaninesarelargelyindependentofthelengthsofthealkylchains.Instead,theyaredeterminedbytheheteroatomsintheterminalringsystemsandthelengthoftheconnectingbridge.Theyhaveextremelyhighextinctioncoefficients,moderatefluorescencequantumyieldsandshortexcitedstatelifetimesinlipidenvironments(~1ns).Thefluorescencespectrumofeachdyeisshownbelow.

YoucanchoosetheFluoroliposome®basedonthetypeofthefluorescentequipmentandfiltersthatyouuseinyourlab.Clodronateliposomescannotbemadefluorescentsimplyduetothepotentialforinaccurateand/oruninterpretabledatabeinggeneratedbylabelledClodrosome®.Formoreinformation,pleaserefertothetechnicalnotesection.

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TechnicalInformation

Fluoroliposome®-DiA

LipidComposition	Concentration(mg/ml)	Concentration(mM)	MolarRatioPercentage
Total	23mg/ml	35.1mM	100
L-alpha-Phosphatidylcholine	18.8	24.3	70
Cholesterol	4.2	10.9	30

FluorescentDye	Excitation/Emission(nm)	Concentration(mg/ml)	Concentration(mM)
4-(4-(Dihexadecylamino)styryl)-N-methylpyridiniumIodide(DiA)	456/590	0.0625	0.0794

BufferandLiposomeSize	Specification
Buffer	PhosphateBufferedSaline
pH	7.4
LiposomeSize	1.5-2µm

TechnicalNotes

• TheissuewithfluorescentClodrosome®hastodowiththepotentialforinaccurateand/oruninterpretabledatabeinggeneratedbylabelledClodrosome®.WhenClodrosome®inducesmacrophageapoptosis,thefluorescentlipidincorporatedintotheClodrosome®isdisruptedandmetabolizedinthephagolysosomewillbedispersedamongtheresidualapoptoticbodieswhicharesubsequentlyphagocytosedbyothermacrophages.Therefore,fluorescentlipidsmaybedetectedinphagocyticcellswhichneverphagocytosedClodrosome®especiallywhenFACSorfluoroscopyareutilizedtodetectfluorescentcells(FACS)orfluorescencelevelsinatissuehomogenate(fluoroscopy).Anotherpotentialartifactarisesfromfluorescentlipidremainingintheextracellular“garbage”,whichhasnotyetbeenclearedbyotherphagocytes,generatingahighbackgroundfluorescence.However,experiencedconfocalmicroscopistmaybeabletodifferentiatebetweenthepunctatefluorescence,resultingfromfluorescentintactliposomesversusthemorediffusefluorescencecharacteristicofdisruptedliposomesandsomehavesuccessfullyusedfluorescentclodronateliposomestovisualizethecellularlocationoftheseliposomesbyconfocalmicroscopyinvivo[1].Afurthercomplicatingfactoristhatpublisheddatavarieswidelyastoexactlywhenclodronateliposomesbegintoinduceapoptosisinmacrophages.Mönkönnnen etal.showthatmacrophagedeathismeasurablewithinthefirsthourafterclodronateliposometreatmentonRAW264cellsinvitro[2],whileothershavereportednosignsofmacrophageapoptosisuntilseveralhoursaftertreatmentinvivo.ThevariABIlityinthedataislikelyduetodifferentLiposomalformulationsofclodronateaswellasthevastlydifferentexperimentalconditions.Therefore,aswithmostBIOLOGicalstudies,especiallythoseinvolvingliposomes,theamountoftimebetweentreatingtheanimalorcellswithclodronateliposomesandtheonsetofapoptosiswillneedtobeestablishedineachexperimentalmodel.IfthenatureoftheresearchdemandsthatClodrosome®betrackedratherthanthecontrol,EncapsulacanprovideDiI-labelledClodrosome®uponrequest,andassumingthattheClodrosome®distributioncandefinitivelybeassessedpriortotheonsetofapoptosis,clearandvaliddataonthebiodistributionoffluorescentClodrosome®shouldbeobtainable.Still,formostpurposes,Fluoroliposome®(fluorescentcontrolliposomes)willprovidetherequireddatawithfarfewerpotentialartifacts.
• Whenmonitoringmonocyteuptakeinvivoinnormalanimals,thecirculatingmonocytesmay“disappear”orshowreducedcountswithinthefirst2hpost-injectionduetomarginationofthemonocytespost-liposomephagocytosis.Thesecellswillre-enterthecirculationwithinafewhours.Sunderkötter etal.demonstratethisphenomenonanddiscussthebehaviorindetail.Alsoconsiderthatcirculatingmonocyteshavealifetimeofabout24hsolabeledmonocyteswillbecontinuallyleavingthecirculation,eveninnormalanimals,duetoagingofthemonocytes[3].
• Liposomesmaysettlewhenleftundisturbedformorethanafewhours.Immediatelypriortouse,inordertoensureahomogeneousliposomesUSPension,slowlyinvertthevialseveraltimesuntilthesuspensionappearshomogeneousbyvisualinspection.Vigorousorerraticshakingwillnotdamagetheliposomesbutmayinducefoamingandbubbleformationmakingitmoredifficulttoaccuratelymeasurethedesireddosage.
• Ifthepersonnelperformingintravenousinjectionsarenotexperiencedinorfamiliarwith,precautionsforinjectinglargervolumes(~10%animalweightinml),viscousliquidsorparticulatesuspensions,considerhavingextraanimalsavailableincaseseriousinjection-relatedadverseeventsoccur.Dosecontrolanimalsfirsttobecomefamiliarwithlargevolumeinjections.
• Whendosingintravenously,usestandardprecautionsfordosinglargervolumestoanimalsincludingthefollowing:a)Warmproducttoroomtemperaturepriortodosing.b)Ensurethatallairbubblesareremovedfromthesyringepriortodosing;intravenousinjectionofairbubblesmayresultinairemboliwhichcankillorseriouslyinjureanimals.c)Injectproductataslow,steadyrateofnomorethan1ml/min;decreaseinfusionrateifanimalsdisplayanyatypicalreactionssuchasunusualagitation.
• Infusion-relatedadversereactionsusuallyinvolvetheanimalgaspingforairorotherseizure-likemovements.Animalsoftenrecoverwithnoapparentpermanentinjury,butanypotentialeffectsonexperimentalresultsmustbeassessedbytheresearcher.
• Liposomesshouldbekeptat4°CandNEVERbefrozen.

Dosage

Appearance

Fluoroliposome®-DiAisayellowliquidsuspensionmadeoflargemicronsizemultilamellarliposomes.Duetotheirlargesize,someliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,Fluoroliposome®-DiAwillphaseseparateandformpelletsinthebottomofthevial,leavingaclearsolutionontop.Therefore,thevialshouldbeshakentoformahomogeneoussolutionpriortouse.

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Ordering/ShippingInformation

• Allliposomebasedformulationsareshippedonblueiceat4°Cininsulatedpackagesusingovernightshippingorinternationalexpressshipping.
• LiposomesshouldNEVERbefrozen.Icecrystalsthatforminthelipidmembranecanrupturethemembrane,changethesizeoftheliposomesandcausetheencapsulateddrugtoleakout.Liposomesinliquidformshouldalwaysbekeptintherefrigerator.
• ClientswhoorderfromoutsideoftheUnitedStatesofAmericaareresponsIBLefortheirgovernmentimporttaxesandcustomspaperwork.EncapsulaNanoSciencesisNOTresponsibleforimportationfeestocountriesoutsideoftheUnitedStatesofAmerica.
• WestronglyencouragetheclientsinJapan,Korea,TaiwanandChinatoorderviaadistributor.Toughcustomsclearanceregulationsinthesecountrieswillcausedelayincustomclearanceoftheseperishableformulationsifordereddirectlythroughus.Distributorscaneasilyclearthepackagesfromcustoms.Toseethelistofthedistributorsclickhere.
• ClientsorderingfromuniversitiesandresearchinstitutesinAustraliashouldkeepinmindthattheliposomeformulationsaremadefromsyntheticmaterialandtheformulationsdonotrequirea“permittoimportquarantinematerial”.LiposomesareNOTbiologicalproducts.
• Ifyouwouldlikeyourinstitute’sFedExorDHLaccounttobechargedforshipping,thenpleaseprovidetheaccountnumberatthetimeofordering.
• EncapsulaNanoScienceshasnocontroloverdelaysduetoinclementweatherorcustomsclearancedelays.YouwillreceiveaFedExorDHLtrackingnumberonceyourorderisconfirmed.ContactFedExorDHLinadvanceandmakesurethatthepaperworkforcustomsisdoneontime. AllsubsequentshippinginquiriesshouldbedirectedtoFederalExpressorDHL.

StorageandShelfLife

Storage

Fluoroliposome®productsshouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.ENSisnotresponsibleforresultsgeneratedbyfrozenproduct.

ShelfLife

Fluoroliposome®productsaremadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin60daysofthemanufacturingdate.

ReferencesandbackgroundreADIng

1.PolflietMM,GoedePH,vanKesteren-HendrikxEM,vanRooijenN,DijkstraCD,vandenBergTK.Amethodfortheselectivedepletionofperivascularandmeningealmacrophagesinthecentralnervoussystem.J.Neuroimmunol.2001Jun1;116(2):188–95.

2.MönkkönenJ,LiukkonenJ,TaskinenM,HeathTD,UrttiA.Studiesonliposomeformulationsforintra-articulardeliveryofclodronate.JournalofControlledRelease.1995Aug;35(2–3):145–54.

3.SunderkötterC,NikolicT,DillonMJ,vanRooijenN,StehlingM,DrevetsDA,LeenenP.SubpopulationsofMouseBloodMonocytesDifferinMaturationStageandInflammatoryResponse.JImmunol.2004Apr1;172(7):4410–7.

4.NagaiH,KuwahiraI,SchwenkeDO,TsuchimochiH,NaraA,OguraS,SonobeT,InagakiT,FujiiY,YamaguchiR,WingenfeldL.Pulmonarymacrophagesattenuatehypoxicpulmonaryvasoconstrictionviaβ3AR/iNOSpathwayinratsexposedtochronicintermittenthypoxia.PLoSOne.2015Jul1;10(7):e0131923.

5.ZhuY,SoderblomC,KrishnanV,AshbaughJ,BetheaJR,LeeJK.Hematogenousmacrophagedepletionreducesthefibroticscarandincreasesaxonalgrowthafterspinalcordinjury.Neurobiologyofdisease.2015Feb28;74:114-25.

6.YunMH,DavaapilH,BrockesJP.Recurrentturnoverofsenescentcellsduringregenerationofacomplexstructure.Elife.2015;4:e05505.

7.ArwertEN,HarneyAS,EntenbergD,WangY,SahaiE,PollardJW,CondeelisJS.AUnidirectionalTransitionfromMigratorytoPerivascularMacrophageIsRequiredforTumorCellIntravasation.Cellreports.2018May1;23(5):1239-48.