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AntibodiesOnline/CytoSelect™ 24Well Cell Migration Assay (5 μm, Fluorometric Format)/ABIN2344845/5188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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AntibodiesOnline/CytoSelect™ 24Well Cell Migration Assay (5 μm, Fluorometric Format)/ABIN2344845/5

Reactivity
Mammalian
Application
CellularAssay(CA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
BrandCytoSelect™
SampleTypeSerum,CellSamples
AnalyticalMethodQuantitative
DetectionMethodFluorometric
CharacteristicsCytoSelect™CellMigrationAssayKitutilizespolycarbonatemembraneinserts(5μmporesize)toassaythemigratorypropertiesofcells.ThekitdoesnotrequireyoutoprelabelthecellswithCalceinAMorremovenon-migratorycells(i.e.cottonswabbing).Anymigratorycellsarefirstdissociatedfromthemembrane,thenlysedanddetectedbythepatentedCyQuant®GRDye(Invitrogen).CytoSelect™CellMigrationAssayKitprovidesarobustsystemforthequantitativedeterminationofcellmigration.ThisTrialSizekitcontainssufficientreagentsfortheevaluationof4samples.The5μmporesizeisoptimalformonocyteandmacrophagecellmigration.However,inthecaseofepithelialandfibroblast,alargerporesize(8μm)isrecommended.Forneutrophilchemotaxis,asmallerporesize(3μm)isrecommended.TheCytoSelect™CellMigrationAssayKitcontainsPETmembraneinserts(5μmporesize)ina24-wellplate.Themembraneservesasabarriertodiscriminatemigratorycellsfromnon-migratorycells.Migratorycellsareabletoextendprotrusionstowardschemoattractants(viaactincytoskeletonreorganization)andultimatelypassthroughtheporesofthepolycarbonatemembrane.ThesemigratorycellsarethendissociatedfromthemembraneandsubsequentlydetectedbythepatentedCyQuant®GRDye(Invitrogen).2
Components
  1. 24-wellMigrationPlate:One24-wellplatecontaining4cellcultureinserts(5μmporesize)
  2. CellDetachmentSolution:One10mLbottle
  3. 4XLysisBuffer:One2mLtube
  4. CyQuant®GRDye:One10μLtube
  5. Forceps:Oneeach
Materialnotincluded
  1. Migratorycelllines
  2. Cellculturemedium
  3. Serumfreemedium,suchasDMEMcontaining0.5%BSA,2mMCaCl2and2mMMgCl2
  4. Cellcultureincubator(37°C,5%CO2atmosphere)
  5. Lightmicroscope
  6. 96-wellplatesuitableforafluorescenceplatereader
  7. Fluorescenceplatereader
BackgroundCellmigrationisahighlyintegrated,multistepprocessthatorchestratesembryonicmorphogenesis,tissuerepairandregeneration.Itplaysapivotalroleinthediseaseprogressionofcancer,mentalretardation,atherosclerosis,andarthritis.Theinitialresponseofacelltoamigration-promotingagentistopolarizeandextendprotrusionsinthedirectionoftheattractant,theseprotrusionscanconsistoflarge,broadlamellipodiaorspike-likefilopodia.Ineithercase,theseprotrusionsaredrivenbyactinpolymerizationandcanbestabilizedbyextracellularmatrix(ECM)adhesionorcell-cellinteractions(viatransmembranereceptors).
ApplicationNotesOptimalworkingdilutionshouldbedeterminedbytheinvestigator.
Comment

  • Fullyquantifychemotaxiswithnomanualcellcounting
  • Measurechemotaxisinlessthan6hourswithmostcelltypes
  • Membraneinsertsareuncoatedtoallowusewithanychemoattractant

AssayProcedure
  1. Understerileconditions,allowthe24-wellmigrationplatetowarmupatroomtemperaturefor10 minutes.
  2. Prepareacellsuspensioncontaining0.5-5.0x106cells/mLinserumfreemedia.Agentsthatinhibitorstimulatecellmigrationcanbeaddeddirectlytothecellsuspension.
  3. Add0.5 mLofmediacontaining10 %fetalbovineserumordesiredchemoattractant(s)tothelowerwellofthemigrationplate.
  4. Add100μLofthecellsuspensionsolutiontotheinsideofeachinsert.
  5. Incubatefor1-24hoursinacellcultureincubator.
  6. Carefullyaspiratethemediafromtheinsideoftheinsert.Transfertheinserttoacleanwellcontaining400μLofCellDetachmentSolution.Incubate30 minutesat37 °C.Note:Retainthemediuminthe24-wellmigrationplatethatcontainschemoattractant(s)andcellsthatmigratedthroughthemembraneandintothemedium.4
  7. Completelydislodgethecellsfromtheundersideofthemembranebygentlytiltingtheinsertseveraltimesinthedetachmentsolution.Removeanddiscardtheinsert.
  8. Transfer400μLofthe0.5 mLmediumsolutioncontainingmigratorycells(step5)tothewellthatcontains400μLofCellDetachmentSolutionforthesamemigrationassaysample(step7).Mixwell,transfer180μLofthemixturetoa96-wellplate.Note:Thisstepcombinescellsthatmigratedthroughthemembraneandintothemedium,andmigratorycellsdetachedfromthebottomsideofthemembranebyCellDetachmentSolution.
  9. Preparesufficient4XLysisBuffer/CyQuant®GRdyesolutionforallsamplesbydilutingthedye1:75in4XLysisBuffer(forexample,add5μLdyeto370μLof4XLysisBuffer).
  10. Add60μLof4XLysisBuffer/CyQuant®GRdyesolutiontoeachwellofthe96-wellplatecontainingmigratorycells.Incubate20 minutesatroomtemperature.
  11. Transfer200μLofthemixturea96-wellplatesuitableforfluorescencemeasurement.Readfluorescencewithafluorescenceplatereaderat480nm/520nm.
RestrictionsForResearchUseonly
Storage4°C
StorageCommentStoreallcomponentsat4°C.
Productcitedin:Yao,Abe,Kawasaki,Akbar,Matsuura,Onji,Hiasa:"CharacterizationofLiverMonocyticMyeloid-DerivedSuppressorCellsandTheirRoleinaMurineModelofNon-AlcoholicFattyLiverDisease."in:PLoSONE,Vol.11,Issue2,pp.e0149948,2016(PubMed).

Stranahan,Hao,Dey,Yu,Baban:"Blood-brainbarrierbreakdownpromotesmacrophageinfiltrationandcognitiveimpairmentinleptinreceptor-deficientmice."in:Journalofcerebralbloodflowandmetabolism:officialjournaloftheInternationalSocietyofCerebralBloodFlowandMetabolism,Vol.36,Issue12,pp.2108-2121,2016(PubMed).

Du,Wong,Song,Shen,Mori,Rotllan,Price,Dobrian,Meng,Kleinstein,Fernandez-Hernando,Goldstein:"Age-associatedvascularinflammationpromotesmonocytosisduringatherogenesis."in:Agingcell,Vol.15,Issue4,pp.766-77,2016(PubMed).

Lei,Dong,Wu,Dong,Li,Chan,Zhou,Yuan:"LentiviralDeliveryofSmallHairpinRNATargetingConnectiveTissueGrowthFactorBlocksProfibroticSignalinginTenonsCapsuleFibroblasts."in:Investigativeophthalmology&visualscience,Vol.57,Issue13,pp.5171-5180,2016(PubMed).

Yu,Liu,Zong,Yu,Yang,Liang,Ye,Nong,Jia,Lu,Han:"MesenchymalstemcellswithSirt1overexpressionsuppressbreasttumorgrowthviachemokine-dependentnaturalkillercellsrecruitment."in:Scientificreports,Vol.6,pp.35998,2016(PubMed).

Choi,Choi,Park,Lee,Kang,Lee,Yeom,Jung,Kim,Lee,Kang:"CytosolicHsp60orchestratesthesurvivalandinflammatoryresponsesofvascularsmoothmusclecellsininjuredaorticvessels."in:Cardiovascularresearch,Vol.106,Issue3,pp.498-508,2015(PubMed).

Sa,Li,Li,Guo:"TIMP-1Induces?-SmoothMuscleActininFibroblaststoPromoteUrethralScarFormation."in:Cellularphysiologyandbiochemistry:internationaljournalofexperimentalcellularphysiology,biochemistry,andpharmacology,Vol.35,Issue6,pp.2233-43,2015(PubMed).

Deng,Feng,Deng:"TIPE2MediatestheSuppressiveEffectsofShikoninonMMP13inOsteosarcomaCells."in:Cellularphysiologyandbiochemistry:internationaljournalofexperimentalcellularphysiology,biochemistry,andpharmacology,Vol.37,Issue6,pp.2434-43,2015(PubMed).

Lee,Shynlova,Lye:"Stretch-inducedhumanmyometrialcytokinesenhanceimmunecellrecruitmentviaendothelialactivation."in:Cellular&molecularimmunology,Vol.12,Issue2,pp.231-42,2015(PubMed).

Liu,Dong,Jiang,Wu,Chan,Zhang,Li,Qin,Sun:"MicroRNA-10bdownregulationmediatesacuterejectionofrenalallograftsbyderepressingBCL2L11."in:Experimentalcellresearch,2015(PubMed).

Wu,Ding,Cao,Zhang:"FBXL5inhibitsmetastasisofgastriccancerthroughsuppressingSnail1."in:Cellularphysiologyandbiochemistry:internationaljournalofexperimentalcellularphysiology,biochemistry,andpharmacology,Vol.35,Issue5,pp.1764-72,2015(PubMed).

Ying,Sun,He:"BoneMorphogeneticProtein-7InhibitsEMT-AssociatedGenesinBreastCancer."in:Cellularphysiologyandbiochemistry:internationaljournalofexperimentalcellularphysiology,biochemistry,andpharmacology,Vol.37,Issue4,pp.1271-8,2015(PubMed).

Jelacic,Chabot,Bozue,Tobery,West,Moody,Yang,Oppenheim,Friedlander:"ExposuretoBacillusanthraciscapsuleresultsinsuppressionofhumanmonocyte-deriveddendriticcells."in:Infectionandimmunity,Vol.82,Issue8,pp.3405-16,2014(PubMed).

Cizkova,Devaux,LeMarrec-Croq,Franck,Slovinska,Blasko,Rosocha,Spakova,Lefebvre,Fournier,Salzet:"Modulationpropertiesoffactorsreleasedbybonemarrowstromalcellsonactivatedmicroglia:aninvitrostudy."in:Scientificreports,Vol.4,pp.7514,2014(PubMed).

Kiss,Mollenhauer,Walmsley,Kirchberg,Radhakrishnan,Niemietz,Dudda,Steinert,Whyte,Carmeliet,Mazzone,Weitz,Schneider:"LossoftheoxygensensorPHD3enhancestheinnateimmuneresponsetoabdominalsepsis."in:Journalofimmunology(Baltimore,Md.:1950),Vol.189,Issue4,pp.1955-65,2012(PubMed).

Shynlova,Tsui,Dorogin,Lye:"Monocytechemoattractantprotein-1(CCL-2)integratesmechanicalandendocrinesignalsthatmediatetermandpretermlabor."in:Journalofimmunology(Baltimore,Md.:1950),Vol.181,Issue2,pp.1470-9,2008(PubMed).

Rayner,Chen,McNulty,Simard,Zhao,Wells,deBelleroche,OBrien:"Extracellularreleaseoftheatheroprotectiveheatshockprotein27ismediatedbyestrogenandcompetitivelyinhibitsacLDLbindingtoscavengerreceptor-A."in:Circulationresearch,Vol.103,Issue2,pp.133-41,2008(PubMed).


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