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AntibodiesOnline/CytoSelect™ 96well Collagen Cell Invasion Assay, Fluorometric/ABIN2344868/96 test188bio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™ 96well Collagen Cell Invasion Assay, Fluorometric/ABIN2344868/96 test
Reactivity
Mammalian
Application
CellularAssay(CA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheCytoSelect™96-wellCollagenCellInvasionAssayKitcontainspolycarbonatemembraneinserts(8μmporesize)ina96-wellplate.TheuppersurfaceoftheinsertmembraneiscoatedwithauniformlayerofdriedBovineTypeICollagenmatrix.Thiscollagenmatrixlayerservesasabarriertodiscriminateinvasivecellsfromnon-invasivecells.Invasivecellsareabletodegradethecollagenmatrixlayer,andultimatelypassthroughtheporesofthepolycarbonatemembrane.Finally,theseinvadedcellsarethendissociatedfromthemembraneandsubsequentlydetectedbythepatentedCyQuant®GRDye(Invitrogen).2
BrandCytoSelect™
SampleTypeSerum,CellSamples
AnalyticalMethodQuantitative
DetectionMethodFluorometric
CharacteristicsCytoSelect™96-wellCollagenCellInvasionAssayKitutilizesBovineTypeICollagen-coatedinsertstoassaytheinvasivepropertiesoftumorcells.ThekitdoesnotrequireyoutoprelabelthecellswithCalceinAMorremovenon-invadedcells(i.e.cottonswabbing).Anyinvadedcellsarefirstdissociatedfromthemembrane,thenlysedanddetectedwithCyQuant®GRDye.TheCytoSelect™96-wellCollagenCellInvasionAssayKitprovidesarobustsystemforthequantitativedeterminationofcellinvasion.Itcontainssufficientreagentsfortheevaluationof96samples.
Components
  1. 96-wellCollagenInvasionPlate:Onesterile96-wellplatecontainingcollagen-coatedinserts(seeFigure1forcomponents)
  2. 96-wellCellHarvestingTray:One96-welltray
  3. CellDetachmentSolution:One20mLbottle
  4. 4XLysisBuffer:One10mLbottle
  5. CyQuant®GRDye:One75μLtube
Materialnotincluded
  1. Invasivecelllines
  2. Cellculturemedium
  3. Serumfreemedium,suchasDMEMcontaining0.5%BSA,2mMCaCl2and2mMMgCl2
  4. Cellcultureincubator(37°C,5%CO2atmosphere)
  5. Lightmicroscope
  6. 96-wellmicrotiterplate
  7. Microtiterplatereader3TopPlateCoverMiddleInvasionPlateMembraneChamberBottomFeederTray:Componentsofthe96-wellCollagenCellInvasionPlate.
BackgroundTheabilityofmalignanttumorcellstoinvadenormalsurroundingtissuecontributesinlargeparttothesignificantmorbidityandmortalityofcancers.Invasivenessrequiresseveraldistinctcellularfunctionsincludingadhesion,motility,detachment,andextracellularmatrixproteolysis.Metastaticcellsproducemanyproteolyticenzymes(e.g.lysosomalhydrolysates,Collagenases,plasminogenactivators)whiletheexpressionofcertaincellsurfaceproteasereceptorsisalsoincreased.
ApplicationNotesOptimalworkingdilutionshouldbedeterminedbytheinvestigator.
Comment

  • Fullyquantifycellinvasionwithnomanualcellcounting
  • PlateinsertsareprecoatedwithCollagenIgellayer

PlatePre-coated
AssayProcedure
  1. Understerileconditions,allowthecollageninvasionplatetowarmupatroomtemperaturefor10 minutes.
  2. Rehydratethecollagenlayerofthemembraneinsertsbyadding125μLofwarm,serum-freemediatotheinnercompartment.Incubateatroomtemperaturefor30 minutes.
  3. Prepareacellsuspensioncontaining0.2-2.0x106cells/mLinserumfreemedia.Agentsthatinhibitorstimulatecellinvasioncanbeaddeddirectlytothecellsuspension.Note:Overnightstarvationmaybeperformedpriortorunningtheassay.
  4. Carefullyremove100μLoftherehydrationmedium(step2)fromtheinsertswithoutdisturbingthecollagenlayer(leaving25μLinside).4
  5. Understerileconditions,separatethecoverandmembranechamberfromthefeedertray.Add150μLofmediacontaining10 %fetalbovineserumordesiredchemoattractant(s)tothewellsofthefeedertray.
  6. Placethemembranechamberbackintothefeedertray(containingchemoattractantsolution).Ensurenobubblesaretrappedunderthemembrane.
  7. Gentlymixthecellsuspensionfromstep3andadd100μLtothemembranechamber.
  8. Finally,covertheplateandtransfertoacellcultureincubatorfor12-24hours.
  9. Justpriortotheendoftheincubation,pipette150μLofprewarmedCellDetachmentSolutionintowellsoftheclean,96-WellCellHarvestingTray(provided).
  10. Carefullyremovethe96-wellInvasionPlatefromtheincubator.Separatethemembranechamberfromthefeedertray.
  11. Removethecells/mediafromthetopsideofthemembranechamberbyaspiratingorinverting.PlacethemembranechamberintotheCellHarvestingTraycontaining150μLofCellDetachmentSolution(step9).Incubate30 minutesat37 °C.
  12. CompletelydislodgethecellsfromtheundersideofthemembranebygentlytiltingthemembranechamberseveraltimesintheCellDetachmentSolution.
  13. Preparesufficient4XLysisBuffer/CyQuant®GRdyesolutionforallsamplesbydilutingthedye1:75in4XLysisBuffer(forexample,add5μLdyeto370μLof4XLysisBuffer).
  14. Add50μLof4XLysisBuffer/CyQuant®GRdyesolutiontoeachwell(alreadycontaining150μLofCellDetachmentSolution).Incubate20 minutesatroomtemperature.
  15. Transfer150μLofthemixturetoa96-wellplatesuitableforfluorescencemeasurement.Readthefluorescencewithafluorescenceplatereaderat480nm/520nm.5
RestrictionsForResearchUseonly
Storage4°C
StorageCommentStoreallcomponentsat4°C.
Productcitedin:Ohishi,Yoshida,Katori,Migita,Muramatsu,Miyake,Ishikawa,Saiura,Iemura,Natsume,Seimiya:"Tankyrase-BindingProteinTNKS1BP1RegulatesActinCytoskeletonRearrangementandCancerCellInvasion."in:Cancerresearch,Vol.77,Issue9,pp.2328-2338,2017(PubMed).

Ben-David,Ha,Khadka,Jin,Wong,Franke,Golub:"Thelandscapeofchromosomalaberrationsinbreastcancermousemodelsrevealsdriver-specificroutestotumorigenesis."in:Naturecommunications,Vol.7,pp.12160,2016(PubMed).

Ho,Goradia,Russell,Chalk,Milley,Baker,Danks,Slavin,Walia,Crimeen-Irwin,Dickins,Martin,Walkley:"KnockdownofPTHR1inosteosarcomacellsdecreasesinvasionandgrowthandincreasestumordifferentiationinvivo."in:Oncogene,Vol.34,Issue22,pp.2922-33,2015(PubMed).

Ishiba,Nagahara,Nakagawa,Sato,Ishikawa,Uetake,Sugihara,Miki,Nakanishi:"Periostinsuppressioninducesdecorinsecretionleadingtoreducedbreastcancercellmotilityandinvasion."in:Scientificreports,Vol.4,pp.7069,2014(PubMed).

Liu,Ma,Dai,Cai,Yao,Yang,Feng,Deng,Li,Ma,Xin,Lian,Xiang,Zhang,Wang:"Antiproliferative,antiinvasive,andproapoptoticactivityoffolatereceptorα-targetedliposomaldoxorubicininnonfunctionalpituitaryadenomacells."in:Endocrinology,Vol.154,Issue4,pp.1414-23,2013(PubMed).

Ziemann,Hess,Bhuwania,Linder,Kloppenburg,Noegel,Clemen:"CRN2enhancestheinvasivenessofglioblastomacells."in:Neuro-oncology,Vol.15,Issue5,pp.548-61,2013(PubMed).


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