Description
m-Clodrosome®isamultilamellarliposomesUSPensioninwhichClodronateisencapsulatedintheaqueouscompartmentsofthemannosylatedliposomes.m-Encapsome®isformulatedandpreparedidenticallytom-Clodrosome®exceptthatclodronateisnotaddedtotheliposomes.Theliposomesarefilteredthrough2μmpolycarbonatemembranestoensurethatlargerparticles,whichmaybetoxictoanimals,areremovedfromthesuspension.Botharepreparedandpackagedundersterileconditions.Whenanimalsorcellsaretreatedwithm-Clodrosome®,phagocyticcellsrecognizetheliposomesasinvADIngforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosolresultingincelldeath.Non-encapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.m-Encapsome®-controlliposomes-arerecognizedandphagocytosedbythesamemechanismasm-Clodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofcontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
Mannosereceptortargetingbymannosylatedliposomeshasbeendemonstratedforavarietyofmannosylatedlipidconjugatesinavarietyofliposomemorphologiesandcompositionsinseveraldifferentinvitroandinvivomodels.Thereareseveralpublicationsaboutusingahydrophobicderivativeofmannose(4-aminophenylalpha-D-mannopyranoside)ratherthanusingamannosylatedlipidinclodronateliposomes.Thisismainlyduetothehighcostandcomplexityofsynthesizingandconjugatingmannosetolipid.4-aminophenylalpha-D-mannopyranosideiscommerciallyavailableandfarlessexpensivethansynthesizingmannoseconjugatedlipid.
Whymannose?Mannoseisoneofthecarbohydratecomponentsofmanybacterialandviralcellsurfaces,thereforetheever-efficient,highlyredundantimmunesystemhasevolvedmultiplemechanismsforidentifyingpathogensbasedonmannoserecognition.Theanimalandplantkingdomslikewiseutilizecarbohydraterecognitionsignalingmechanismsincludingmannoseresidues.Manypublicationsevaluateothercarbohydratesastargetingmechanismsforvariouscelltypes,howevermannosetargetingtophagocytesappearstobeoneofthemorespecificmechanismsidentifiedtodate.Mammaliancellsurfaceidentificationmoleculesbasedonmannosebinding,suchastheICAMfamilyofleukocyteadhesionmolecules,targettheSIGNfamilyofmannosereceptorstoaccomplishself-recognitioninvivo.
Awell-knownandcitedstudybyUmezawa&Eto [1]demonstratesthatliposomescontainingaminophenylmannosideweremostefficientlyincorporatedintothemousebrainacrossthebloodbrainbarrier.Theradiolabeledliposomesbearingaminophenyl-alpha-D-mannopyranosideweremaximallyincorporatedintothemousebrainafter48hours,whereasinthespleenandliver,theseradioactivitiesweremaximumafter12hours.Thestudiesalsoshowedthatliposomesweremostincorporatedwasglialcellsratherthanneuronalcell.Thesubcellularfractionationstudyindicatesthatmannoselabeledliposomesareincorporatedintolysosomesrichfractionbothinliverandbrain.
Mannosylatedfluorescentliposomes(m-Fluoroliposome®)suitableformacrophagetargetingandtrackingareavailablecontainingfivedifferentfluorescentdyes(DiI,DiO,DiD,DiAandDiR)thatcoverstheentirespectrum.Fluorescentliposomescomeinstandardandmannosylatedform.Formoreinformationsee here.
Clodrosome®andEncapsome®arestandardandnon-mannosylatedreagentsthatareusedfortargetingmacrophagesinorgansandtissuesotherthancentralnervoussystems.Formoreinformationaboutthesereagentseehere.
FormulationInformation
m-Encapsome®ControlLiposomeSuspension
| 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 |
| Mannosylation | Concentration |
|---|---|
| 4-Aminophenyl-alpha-D-mannopyranoside | 9.53mol% |
| BufferandLiposomeSize | Specification |
|---|---|
| Buffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 1.5-2µm |
TechnicalNotes
- Toreachbloodstream-accessIBLe,mannose-receptorpositivecellsoutsidetheliver,asignificantnumberofliposomeswillhavetoescapefirst-passuptakebytheliverandspleen,sothatthetargetcellsareexposedtoahigherconcentrationofmannosylatedliposomesfromtheblood.Onestrategythathasbeenusedtoensurethatliposomesescapetheliverandspleenisknownasreticuloendothelialsystem(RES)blockadeinwhichanimalsarepre-dosedwithasufficientquantityofliposomestotemporarilysaturatethephagocyticcellsoftheblood,liverandspleen,alsoknownasthereticuloendothelialsystem(RES)orthemononuclearphagocytesystem(MPS).Thissufficientquantityisdependentupontheliposometypeandcompositionaswellasthespeciesbeingdosed;thepre-dosedliposomesdonotnecessarilyneedtobethesametypeorcompositionasthetherapeuticordiagnosticliposomesavoidingtheRES.Soonafterthispre-doseisclearedfromthebloodstream(usuallywithinacoupleofhours),theliposomesofinterestaredosed.SincetheRESisinvolvedindigestingthepreviousdoseofliposomes,thesubsequentlydosedliposomeswillremaininthecirculationmuchlongerthusbemuchmorelikelytobindtotheirtargetsiteoutsidetheRESincludingthosephagocyticcellswhichareaccessible,butarenotusuallyexposedtoahigherconcentrationofliposomes.
- WhileRESblockadeisusuallythoughtofassaturatingphagocyticcells,ithasbeenshownthatopsonin-bindingbyliposomesisasaturablephenomenon.Therefore,partofRESblockademayinvolveserumdepletionofcomplementandotheropsoninsknowntocoatliposomes.Inthecurrentapplication,removalorreductionintheconcentrationofsolublemannose-receptorsmayfurtherincreasetheprobABIlityofamannosylatedliposomebeingabletointeractwithmannosereceptorsonthetargetcell.Therefore,ifthegoalistodepleteatargetsubsetofmannose-receptor+cellswhichmaynotnormallybeexposedtoasubstantialnumberofmannosylatedliposomes,pre-dosingwithmannosylatedclodronateliposomes,inordertobothsaturatetheblood,liverandspleenphagocytesandreducetheconcentrationofopsoninsincludingsolublemannosereceptors,shouldincreasethenumberofsubsequentlydosedmannosylatedclodronateliposomesavailabletothistargetsubsethypotheticallyresultinginincreaseduptakeanddepletionbythesetargetedcells.
- WhenanimalsorcellsaretreatedwithClodrosome®,phagocyticcellsrecognizetheliposomesasinvadingforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosolresultingincelldeath.Unencapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.
- Encapsome®controlliposomesarerecognizedandphagocytosedbythesamemechanismasClodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofthecontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
- Theproductmustberemovedfromthevialusingsteriletechnique.Donotuseifsterilityiscompromised.Thisisparticularlyimportantifasinglevialisaccessedmultipletimesoverseveralweeks.Theproductshouldnotbeusedmorethan60daysafterreceipt,evenifunopened.
- Liposomesmaysettlewhenleftundisturbedformorethanafewhours.Immediatelypriortouse,inordertoensureahomogeneousliposomesuspension,slowlyinvertthevialseveraltimesuntilthesuspensionappearshomogeneousbyvisualinspection.Vigorousorerraticshakingwillnotdamagetheliposomesbutmayinducefoamingandbubbleformationmakingitmoredifficulttoaccuratelymeasurethedesireddosage.
- Ifthepersonnelperformingintravenousinjectionsarenotexperiencedinorfamiliarwith,precautionsforinjectinglargervolumes(~10%animalweightinml),viscousliquidsorparticulatesuspensions,considerhavingextraanimalsavailableincaseseriousinjection-relatedadverseeventsoccur.Dosecontrolanimalsfirsttobecomefamiliarwithlargevolumeinjections.
- WithinhoursaftersystemicadmiNISTrationofClodrosome®,animalsbegintoloseimportantcomponentsoftheirimmunesystem.Standardanimalhandlingandhousingprotocolsarenotsuitableforimmunocompromisedanimals.Evenwhensuchprecautionsaretaken,monitorthegeneralhealthofeachanimalforopportunisticinfectionsunrelatedtotheexperimentalprotocol.Thereisnoinherenttoxicitytotheproductattherecommendeddoselevels.
- Whendosingintravenously,usestandardprecautionsfordosinglargervolumestoanimalsincludingthefollowing:a)warmproducttoroomtemperaturepriortodosing;b)ensurethatallairbubblesareremovedfromthesyringepriortodosing.Intravenousinjectionofairbubblesmayresultinairemboliwhichcankillorseriouslyinjureanimals;c)injectproductataslow,steadyrateofnomorethan1ml/min;d)decreaseinfusionrateifanimalsdisplayanyatypicalreactionssuchasunusualagitation.
- Infusion-relatedadversereactionsusuallyinvolvetheanimalgaspingforairorotherseizure-likemovements.Animalsoftenrecoverwithnoapparentpermanentinjury,butanypotentialeffectsonexperimentalresultsmustbeassessedbytheresearcher.
- Liposomesshouldbekeptat4°CandNEVERbefrozen.
Dosage
Appearance
m-Clodrosome®andm-Encapsome®arebothwhitemilkysuspensionsmadeoflargemicrosizemultilamellarliposomes.Duetotheirlargesizesomeliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,m-Encapsome®willphaseseparateandformpelletsinthebottomofthevialleavingaclearsolutionontop.m-Clodrosome®mightdothesameonlynotasseverely.Therefore,bothshouldbegentlyshakennottoformbubblesbuttoformahomogeneoussolutionpriortouse.
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
m-Clodrosome®andm-Encapsome®shouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.Ifthesuspensionisfrozen,clodronatecanbereleasedfromtheliposomesthuslimitingitseffectivenessindepletingmacrophages.ENSisnotresponsibleforresultsgeneratedbyfrozenproduct.
ShelfLife
m-Clodrosome®andm-Encapsome®aremadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin60daysofthemanufacturingdate.
Referencesandbackgroundreading
1.UmezawaFA,EtoY.Liposometargetingtomousebrain:mannoseasarecognitionMarker.Biochemicalandbiophysicalresearchcommunications.1988Jun30;153(3):1038-44.
2.ItoT,IshigamiM,MatsushitaY,HirataM,MatsubaraK,IshikawaT,HibiH,UedaM,HirookaY,GotoH,YamamotoA.SecretedEctodomainofSIGLEC-9andMCP-1SynergisticallyImproveAcuteLiverFailureinRatsbyAlteringMacrophagePolarity.Scientificreports.2017Mar8;7:44043.
3.KanoF,MatsubaraK,UedaM,HibiH,YamamotoA.SecretedEctodomainofSialicAcid‐BindingIg‐LikeLectin‐9andMonocyteChemoattractantProtein‐1SynergisticallyRegenerateTransectedRatPeripheralNervesbyAlteringMacrophagePolarity.STEMCELLS.2017Mar1;35(3):641-53.
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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
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。
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。
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