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®-DiO
| 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) |
|---|---|---|---|
3,3"-DilinoleyloxacarbocyaninePerchlorate(DiO) | 484/501 | 0.0625 | 0.071 |
| 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,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®-DiOisayellowliquidsuspensionmadeoflargemicronsizemultilamellarliposomes.Duetotheirlargesize,someliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,Fluoroliposome®-DiOwillphaseseparateandformpelletsinthebottomofthevial,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.
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
转录的只是插入的片断，怎么会跟模板一样大呢，一般情况下应该比模板小多了。我都是跑电泳，严格来说应该跑变性的；嫌麻烦可以非变性，快速（防止RNA降解）十分钟即可；抛出来的带会有两条，上面大的是模板，下面一条比较亮的是RNA，那么你的转录就 成功了。
PCR第一个步骤如果没记错的话应该叫“变性”， PCR的话是不需要解旋酶的，直接利用DNA的高温变性特性，在高温下，使氢键断开，DNA双链恢复为单链。 （还有解旋酶好像是解除双螺旋的结构，是一种拓扑异构酶，对于氢键的打开没有贡献吧
对于新手来说购买反转录试剂盒是比较理想的，买一个kit看看说明书操作就可以了。推荐两款kit TAKARA和HaiGene，这两款试剂盒都能对RNA样品中的gDNA有效去除，因此对RNA质量的要求不高。TAKARA的RR047A操作方便，反转录温度为37℃，对于大多数试验来讲是满足要求的。HaiGene的D0401操作要多一个步骤，但其反转录温度是55℃（耐高温的反转录酶），提高了反转录温度使得高GC含量、复杂模板、长mRNA的模板都能有效反转录，因此其反转录效率更高，更能够获得样本中基因的真实表达量。如果后续试验是RealTime PCR、ORF克隆、高GC含量、或者你的待研究基因结构复杂程度未知，还是选用耐高温的反转录酶更理想。对于反转录高手来说，直接购买反转录酶、再购买Rnase Inhibitor自己配制反转录体系就可以了。对于样本量大的课题组来讲，相对还是比较经济的。选择反转录酶时仅需要考虑是否需要耐高温的酶，来克服目的基因的复杂结构就可以了。PROMEGA、TAKARA、HaiGene、 TRANSGEN等品牌的反转录酶性价比还都是不错的。Life、NEB的也不错，不过性价比一般，不一一解释了。
首先，不否认它比大多数国产的试剂盒做得好，提取的纯度、方便度和量，都还很不错。
其次，它是日本的。so我觉得实验室用用天根的，就非常好了。拒绝日货，从实验室做起。
