Description
Clodrosome®isamultilamellarliposomesUSPensioninwhichClodronateisencapsulatedintheaqueouscompartmentsoftheliposomes.Encapsome®isformulatedandpreparedidenticallytoClodrosome®exceptthatclodronateisnotaddedtotheliposomes.Theliposomesarefilteredthrough2μmpolycarbonatemembranestoensurethatthelargerparticles,whichmaybetoxictoanimals,areremovedfromthesuspension.Botharepreparedandpackagedundersterileconditions.WhenanimalsorcellsaretreatedwithClodrosome®,phagocyticcellsrecognizetheliposomesasinvADIngforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosol,resultingincelldeath.Non-encapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.
Controlliposomes(Encapsome®)arerecognizedandphagocytosedbythesamemechanismasClodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofcontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
MacrophagedepletionkitiscomposedofonevialofClodrosome®andonevialof Encapsome®.Asanexample,a5-mlkitiscomposedof 5mlClodrosome®and5mlEncapsome®.
m-Clodrosome®andm-Encapsome®aremannosylatedreagentsthatarespecificallyformulatedtoefficientlytargetthemacrophagesincentralnervoussystemsandmacrophagesthatcontainmoremannosereceptors.Formoreinformationaboutthesereagentseehere.
Fluorescentliposomes(Fluoroliposome®)suitableformacrophagetargetingandtrackingareavailablecontainingfivedifferentfluorescentdyes(DiI,DiO,DiD,DiAandDiR)thatcoverstheentirespectrum.Fluorescentliposomescomeinstandardandmannosylatedform.Formoreinformationsee here.
TechnicalInformation
Clodrosome®LiposomalClodronateSuspension
| 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 |
| EncapsulatedDrug | Concentration |
|---|---|
| Clodronate((Dichloro-phosphono-methyl)phosphonate),DisodiumSalt | 18.4*mM |
| *Dependingonthetypeoftheclodronatesalt,itsconcentration(mg/ml)varies.Iftetrahydratesaltisused,theconcentrationoftheencapsulateddrugwillbe~7mg/ml,andifanon-hydratedsaltisused,theconcentrationwillbe~5mg/ml. | |
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 |
| BufferandLiposomeSize | Specification |
|---|---|
| Buffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 1.5-2µm |
Dosage
TechnicalNotes
- 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;decreaseinfusionrateifanimalsdisplayanyatypicalreactionssuchasunusualagitation.
- Infusion-relatedadversereactionsusuallyinvolvetheanimalgaspingforairorotherseizure-likemovements.Animalsoftenrecoverwithnoapparentpermanentinjury,butanypotentialeffectsonexperimentalresultsmustbeassessedbytheresearcher.
- Liposomesshouldbekeptat4°CandNEVERbefrozen.
Appearance
Clodrosome®andEncapsome®arebothwhitemilkysuspensionsmadeoflargemicrosizemultilamellarliposomes.Duetotheirlargesize,someliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,Encapsome®willphaseseparateandformpelletsinthebottomofthevialleavingaclearsolutionontop.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
Clodrosome®andEncapsome®shouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.Ifthesuspensionisfrozen,clodronatecanbereleasedfromtheliposomesthuslimitingitseffectivenessindepletingmacrophages.ENSisnotresponsibleforresultsgeneratedbyfrozenproduct.
ShelfLife
Clodrosome®andEncapsome®aremadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin60daysofthemanufacturingdate.
Referencesandbackgroundreading
1.HinsonSR,CliftIC,LuoN,KryzerTJ,LennonVA.Autoantibody-inducedinternalizationofCNSAQP4waterchannelandEAAT2glutamatetransporterrequiresastrocyticFcreceptor.ProceedingsoftheNationalAcademyofSciences.2017May23;114(21):5491-6.
2.DhupkarP,GordonN,StewartJ,KleinermanES.Anti‐PD‐1therapyredirectsmacrophagesfromanM2toanM1phenotypeinducingregressionofOSlungmetastases.CancerMedicine.2018May7.
3.XiongY,PageJC,NarayananN,WangC,JiaZ,YueF,ShiX,JinW,HuK,DengM,ShiR.Peripheralneuropathyandhindlimbparalysisinamousemodelofadipocyte-specificknockoutofLkb1.EBioMedicine.2017Oct1;24:127-36.
4.CriderA,FengT,PandyaCD,DavisT,NairA,AhmedAO,BabanB,TureckiG,PillaiA.Complementcomponent3areceptordeficiencyattenuateschronicstress-inducedmonocyteinfiltrationanddepressive-likebehavior.Brain,behavior,andimmunity.2018Mar5.
5.KocherT,AsslaberD,ZaborskyN,FlenadyS,DenkU,ReinthalerP,AblingerM,GeisbergerR,BauerJW,SeiffertM,HartmannTN.CD4+Tcells,butnotnon-classicalmonocytes,aredispensableforthedevelopmentofchroniclymphocyticleukemiaintheTCL1-tgmurinemodel.Leukemia.2016Jun;30(6):1409.
6.ZhuZ,DingJ,MaZ,IwashinaT,TredgetEE.Systemicdepletionofmacrophagesinthesubacutephaseofwoundhealingreduceshypertrophicscarformation.WoundRepairandRegeneration.2016Jul1;24(4):644-56.
7.HaqueMR,LeeDY,AhnCH,JeongJH,ByunY.Localco-deliveryofpancreaticisletsandliposomalclodronateusinginjectablehydrogeltopreventacuteimmunereactionsinatype1diabetes.Pharmaceuticalresearch.2014Sep1;31(9):2453-62.
8.MayoL,CunhaAP,MadiA,BeynonV,YangZ,AlvarezJI,PratA,SobelRA,KobzikL,LassmannH,QuintanaFJ.IL-10-dependentTr1cellsattenuateastrocyteactivationandamelioratechroniccentralnervoussysteminflammation.Brain.2016May31;139(7):1939-57.
9.KermanizadehA,ChauchéC,BalharryD,BrownDM,KanaseN,BoczkowskiJ,LanoneS,StoneV.TheroleofKupffercellsinthehepaticresponsetosilvernanoparticles.Nanotoxicology.2014Aug31;8(sup1):149-54.
10.NandiB,ShapiroM,SamurMK,PaiC,FrankNY,YoonC,PrabhalaRH,MunshiNC,GoldJS.StromalCCR6drivestumorgrowthinamurinetransplantablecoloncancerthroughrecruitmentoftumor-promotingmacrophages.Oncoimmunology.2016Aug2;5(8):e1189052.
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逆转录（reverse transcription）是以RNA为模板合成DNA的过程，即RNA指导下的DNA合成。此过程中，核酸合成与转录（DNA到RNA）过程与遗传信息的流动方向（RNA到DNA）相反，故称为逆转录。逆转录过程是RNA病毒的复制形式之一，需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现，是对中心法则的重要修正和补充。人们通过体外模拟该过程，以样本中提取的mRNA为模板，在逆转录酶的作用下，合成出互补的cDNA，构建cDNA文库，并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
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大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊，求推荐！
