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.
TechnicalInformation
Fluoroliposome®-DiD
| 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) |
|---|---|---|---|
1,1"-Dioctadecyl-3,3,3",3"-Tetramethylindodicarbocyanine,4-ChlorobenzenesulfonateSalt(DiD) | 644/665 | 0.0625 | 0.065 |
| BufferandLiposomeSize | Specification |
|---|---|
| Buffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 1.5-2µm |
TechnicalNotes
- TECHNICALNOTES
- TheissuewithfluorescentClodrosome®hastodowiththepotentialforinaccurateand/oruninterpretabledatabeinggeneratedbylabelledClodrosome®.WhenClodrosome®inducesmacrophageapoptosis,thefluorescentlipidincorporatedintotheClodrosome®isdisruptedandmetabolizedinthephagolysosomewillbedispersedamongtheresidualapoptoticbodieswhicharesubsequentlyphagocytosedbyothermacrophages.Therefore,fluorescentlipidsmaybedetectedinphagocyticcellswhichneverphagocytosedClodrosome®especiallywhenFACSorfluoroscopyareutilizedtodetectfluorescentcells(FACS)orfluorescencelevelsinatissuehomogenate(fluoroscopy).Anotherpotentialartifactarisesfromfluorescentlipidremainingintheextracellular“garbage”,whichhasnotyetbeenclearedbyotherphagocytes,generatingahighbackgroundfluorescence.However,experiencedconfocalmicroscopistmaybeabletodifferentiatebetweenthepunctatefluorescence,resultingfromfluorescentintactliposomesversusthemorediffusefluorescencecharacteristicofdisruptedliposomesandsomehavesuccessfullyusedfluorescentclodronateliposomestovisualizethecellularlocationoftheseliposomesbyconfocalmicroscopy invivo [1].Afurthercomplicatingfactoristhatpublisheddatavarieswidelyastoexactlywhenclodronateliposomesbegintoinduceapoptosisinmacrophages.Mönkönnnen etal. showthatmacrophagedeathismeasurablewithinthefirsthourafterclodronateliposometreatmentonRAW264cells invitro [2],whilemanyothershavereportednosignsofmacrophageapoptosisuntilseveralhoursaftertreatment invivo.ThevariABIlityinthedataislikelyduetodifferentLiposomalformulationsofclodronateaswellasthevastlydifferentexperimentalconditions.Therefore,aswithmostBIOLOGicalstudies,especiallythoseinvolvingliposomes,theamountoftimebetweentreatingtheanimalorcellswithclodronateliposomesandtheonsetofapoptosiswillneedtobeestablishedineachexperimentalmodel.IfthenatureoftheresearchdemandsthatClodrosome®betrackedratherthanthecontrol,EncapsulacanprovideDiI-labelledClodrosome®uponrequest,andassumingthattheClodrosome®distributioncandefinitivelybeassessedpriortotheonsetofapoptosis,clearandvaliddataonthebiodistributionoffluorescentClodrosome®shouldbeobtainable.Still,formostpurposes,Fluoroliposome®(fluorescentcontrolliposomes)willprovidetherequireddatawithfarfewerpotentialartifacts.
- Whenmonitoringmonocyteuptake invivo innormalanimals,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°Cand NEVER befrozen.
Dosage
Appearance
Fluoroliposome®-DiDisablueliquidsuspensionmadeoflargemicronsizemultilamellarliposomes.Duetotheirlargesize,someliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,Fluoroliposome®-DiDwillphaseseparateandformpelletsinthebottomofthevial,leavingaclearsolutionontop.Therefore,thevialshouldbeshakentoformahomogeneoussolutionpriortouse.
EducationalVideos
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.
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采用抗-HBe抗体包被反应板，加入校准品及被测样本，同时加入定量HBeAg中和抗原，经过振荡孵育,洗板后再加入铕标记的抗-HBe，若标本中抗-HBe浓度高，HBeAg将被大量中和，使最后形成的抗-HBe-HBeAg-铕标记抗-HBe复合物减少。增强液（β－NTA）将标记在抗体上的Eu3+解离到溶液中，Eu3+和增强液中的有效成分形成高荧光强度的螯合物，荧光强度和样本中的抗-HBe浓度成反比。
1、直接竞争，标记抗原，与检测样品中的抗原竞争抗体。
2、间接竞争，标记抗体，固相抗原与液相抗原（样品）竞争标记抗体。
3、定义
间接竞争法的模型：包被抗原，用HRP-抗体与样本一起加入。样本中的Ag与Solid-Ag竞争HRP-Ab，固相吸附的HRP-Ab与样本中的Ag浓度成反比。
直接竞争法的模型：包被抗体，用HRP-抗原与样本一起加入。样本中的Ag与HRP-Ag竞争Solid-Ab，固相吸附的HRP-Ag与样本中的Ag浓度成反比。
4、竞争法的理论基础：是限量抗体。只有在限量抗体基础上，两种抗原才会形成竞争关系。
5、、间接竞争法具备较高灵敏度原因。
直接竞争法里，标记抗原与待测抗原均是液相，与抗体的结合机会是一样的，例如有1份标记抗原与1份待测抗原竞争1份抗体，那么有50%的标记抗原能与抗体结合，所以标记抗原的相对结合率为50%。间接法里，固相抗原与抗体的接触面积较小，固相抗原与待测抗原的结合抗体机会是不平等的，接近顺序饱和法，即只有与待测抗原结合剩余的抗体才会与固相抗原结合，同样有1份固相抗原与1份待测抗原竞争1份抗体时，基本上抗体会被待测抗原中和掉，与固相抗原结合的抗体非常少。固相抗原的相对结合率为0%。因此，间接法的抑制曲线斜率会大于竞争法。
因为抑制率越大则斜率越大，从而灵敏度越大。（假设零管变异5%，以两倍SD为灵敏度限，则为90%相对结合率，则间接法可以在较低的待测抗原浓度达到这一相对结合率，因此灵敏度要高。）
在进行系统放大时，间接法一般可以使用酶标二抗。因为二抗可以针对抗体的多个部位，所以存在放大效应，从而能提高间接法的灵敏度。直接法一般难以进行放大，常用的有生物 素化抗原与酶标亲和素，但模式上似乎不存在放大效应。
6、间接法的高灵敏度难以实现的原因：
双抗体 夹心的免放（IRMA）模式刚出现时，也被模型证明灵敏度优于竞争法的放免（RIA），原因也是较大的斜率，但是IRMA的高灵敏度一直到单抗发展后才得以实现。
