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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如果是用转录出的cDNA做那跟平时就一样了。不需要特别的试剂盒、
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SYBRGreenMix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBRGreen，并且如果你那是ABI或者Stratagene的PCR如果用SYBRGreen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。
一、cDNA第二链的合成:
1. 第一链反应完成后,取2ul一链产物－20℃冰箱中保存，待电泳检测。其余的产物合并，混匀，然后顺序加入下列试剂(promega):
20ul 10×DNA Polymerase I buffer
6ul10mM dNTP(自己配制)
xuldd H2O
1ulRNase H(2U/ul)
10ul DNA Polymerase I(10U/ul)
总体系为200ul;
2. 混匀后,16℃反应2.5小时;
3. 70℃灭活10分钟;
4. 反应完成后,得到200ul cDNA第二链反应体系,将此体系置于冰上;
5．取2ul二链产物，同保存的一链产物一起电泳鉴定。同时上1kb ladder，确定双链的大小范围。
注：一链，二链的电泳图是smear，且二链稍比一链大一些。
二、双链cDNA末端补平:
1. 在第二链反应体系中,顺序加入下列试剂(promega):
6ul 10mM dNTP
2ul T4 DNA Polymerase(8.7U/ul)
2ul BSA(10mg/ml)
2. 稍微离心混匀反应物, 37℃反应至少30分钟,然后75℃灭活10分钟;
3. 加入等体积酚/氯仿/异戊醇,剧烈振荡后,常温下13000g离心5分钟;
4. 离心后,吸取上清于另一1.5ml eppendof管中,加入等体积氯仿,上下颠倒几次混匀后,常温下13000g离心5分钟;
5. 吸取上清至另一eppendof管,加入1/10V3M NaAc(PH5.2)和2.5V预冷的无水乙醇,混匀,-20℃放置过夜以沉淀双链cDNA;
6. 第二日,将昨日沉淀物在4℃,13000g离心60分钟以充分沉淀双链cDNA;
7.离心完毕,弃上清,加入1ml 70%乙醇洗涤沉淀,常温下13000g离心5分钟;
8.离心完毕,弃上清,干燥沉淀至无乙醇气味.
注：第3，第4步可以用PCR 纯化试剂盒代替。
PCR纯化试剂盒操作流程：
1．溶液PE使用前应加入适量体积95％－100％的乙醇，混匀。
2．向200ul二链补平产物中加入5倍体积的buffer PB，混匀。
3．加入spin column中，13000rpm离心1min。
4．加入0.75ml buffer PE，13000rpm离心1min。
5．13000rpm，再离心1min。
6．将spin column放入一新的离心管中，加入50ul buffer EB，静置10min。
7．13000rpm离心2min。
8．加入30ul buffer EB，静置10min。
9．13000rpm离心2min。
10．加入1/10体积3M的NaAc，2.5倍体积无水乙醇，混匀，－20℃沉淀过夜。
大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊，求推荐！
