高级BioMatrix/CytoSoft®Discovery套件//5190-7EA

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¥4000.00
货号:5190-7EA
浏览量:127
品牌:Advanced BioMatrix
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商品描述

ProductDescription

ThisCytoSoft®product,CatalogNumber5190-7EA,hasvariouselasticmoduliofapproximately0.2,0.5,2,8,16,32and64kPain7individual6-wellplates.Thiskitprovidesyouwith1plateofeachstiffness,allowingyoutotesthowyourcellsbehaveacrossabroadrangeofphysiologicallyrelevantstiffness.

Thethicknessofthesiliconegelisuniformwitha~0.5mmthicklayerofsiliconeineachdishthatisfullycompatIBLewithmammaliancellcultures.ThesiliconegelsareactivatedandreadytobindtoapurifiedECM,suchasPureCol®typeIcollagen(#5005)priortocelladdition.Theplatesarepackagedindividuallyandsterilized,providedwith7dishesperpackage.

Therigidityofthesubstratetowhichcellsadherecanhaveaprofoundeffectoncellmorphologyandgeneexpression.CytoSoft®productsprovideatooltoculturecellsonsubstrateswithvariousrigiditiescoveringabroadphysiologicalrange.Onthebottomofeachwell,thereisathinlayerofspeciallyformulatedbiocompatiblesilicone,whoseelasticmodulus(rigidity)iscarefullymeasuredandcertified.ThesurfacesofthegelsinCytoSoft®productsarefunctionalizedtoformcovalentbondswithaminesonproteins.Thischemicalfunctionalizationisstableandthereactiondoesnotrequireacatalyst,facilitatingthecoatingofthegelsurfaceswithmatrixproteinsandplatingcells.

ThesiliconesubstratesofCytoSoft®productsareopticallyclearandhavealowauto-florescence.Thelayerofsiliconeineachdishisfirmlybondedtothebottomofthedish.Unlikehydrogels(suchaspolyacrylamidegels),siliconegelsarenotsusceptibletohydrolysis,donotdrynorswell,areresilientandresistanttotearingorcracking,andtheirelasticmoduli(rigidities)remainnearlyunchangedduringextendedstorageperiods.

CytoSoft®productsaccommodatetheharvestingofcellsusingenzymessuchastrypsinandCollagenase.Thereisnobiochemicalbreakdownofthesubstrateduringorafterenzymetreatment,andtherearenoresidualsofthesubstrateinthesampleretrievedfromaCytoSoft®plate.

Parameter,Testing,andMethodCytoSoft®DiscoveryKit,6-wellPlates#5190-7EA
SterilizationMethodOzone
PlateSize6-WellPlates
QuantityperPackage7Plates
Rigidty(ElasticModulus)0.2,0.5,2,8,16,32,64kPa(exactvaluesprovidedontheCofA)
StorageRoomTemperature
ShelfLifeMinimumof6monthsfromdateofreceipt
PlateSurfaceMaterialFunctionalizedSilicone

GrowthAreaperWell

9.5cm2

TypicalWorkingVolumeperWell

2.0to3.5mL

CellAttachmentAssay

Pass

DirectionsforUse

DownloadthefullPDFversionorcontinuereADIngbelow:

CoatingProcedure

Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.

RemovetheCytoSoft®productfromtheprotectivesleeveinasterilehood.

  1. Prepareextracellularmatrixmaterialbyneutralizinginamine-freebufferpH7.4to7.9(suchas1XDPBS).WedonotrecommendusinggelatinasyourECMprotein.

Note:Pre-warmthecoatingsolutiontoapproximatelyroomtemperaturebeforeuse.

  1. Diluteasneeded,anddispense3mlofsolutionintoeachwelltocoatthesurface.

Note:RecommendeddilutionforPureCol®TypeIcollagenis1:30(~100µg/ml).

Note:Thehydrophobicsurfacerequireslargervolumestocoverthesurfacethandoconventionalplasticdishes

  1. IncubateECMcoatedCytoSoft®atroomtemperature,coveredfor0.5-1hour.
  1. Afterincubation,aspirateanyremainingmaterialandrinsecoatedsurfacesimmediatelytwotimeswithculturemediumorPBS.Leaveabout2.5mlofmediumperwelltokeepsurfacecovered.

Note:DonotallowtheCytoSoft®surfacetobecomedryoncethesurfacehasbeenwetted.

5.Coatedsurfacesarereadyforuse.

StandardharvestingproceduresusedforremovingcellsfromculturewarecanbeemployedforharvestingcellsfromtheCytoSoft®productincludinguseoftrypsin,Accutase®andnon-enzymaticcelldetachmentsolutions.

ProductQ&A

Werecommenda60Xobjectivefortheimagingplates.

Theelasticmodulusismeasuredbytrackingbeadsonthegelsurfaceunderawide-fieldfluorescencemicroscopewithoutanyotherspecializedequipment.Themeasurementshavesmallandsimpletoestimateerrorsandtheirresultsareconfirmedbyconventionaltensiletests.AmastercurveisobtainedrelatingthemixingratiosofthetwocomponentsofSylgard184withtheresultingelasticmoduliofthegels.

Usingprewarmedmediawilldecreasegassolubilityandhelppreventbubblesonthesurfaceofthesilicone.

Thesiliconegelcancrackifthesurfacebecomesdry.

No.Cellmatrixproteinsareattachedtothesurfaceofthegelviacovalentbonding.ItisdifficulttoformanewECMlayeraftercelldetachment.Therearenolongeranyreactivegroupsonthesurfaceofthegelaftertheinitialcellculture.

Thechangeintherefractiveindexofthesiliconedistortsthingsalittle,soDICwillbepossiblebutnotperfect.Also,DICisonlypossibleontheglassbottomimagingplates,notthe6-wellplasticplates.

1.41

Thetwomainissuesare:

1.IneffecientcoatingofthematrixproteinsduetolowpHofthecoatingsolution.

2.Insufficientwashofleftovercellmatrixafterthecoatingprocedure.

No.CytosoftmustbecoatedwithanECMproteinsuchasCollagenorFibronectin.

Thesurfaceisdecoratedwithanhydridefunctionalgroups.

Plasmauseisnotrecommended.PlasmawillinduceformationofahardcrustonthesurfaceandwillchangethemechanicalpropertiesoftheCytoSoft®products.

DonotfreezetheCytoSoft®products.

Whenfrozen,thereisagoodchancethatthesiliconesurfacegetshydrolyzedandabsorbsmoisture,whichwouldinactivatethebindingsitesandmaketheproductnot-usable.

Ifyoufrozetheproductanditisstillfrozen,warmtheCytoSoft®upat60Cwiththebagvented.Thatwillminimizethechanceofthemabsorbingmoisture–butthereisstillachancethattheywillnolongerbefunctional.

UsinggelatinforcoatingCytoSoftisoftenproblematicbecausegelatinoftenhaslowmolecularweightimpuritiesthatblockbindingsitesontheactivatedsurfaceofthesilicone.WerecommendusinghighlypurifiedECM"sinstead.

ProductCellAssay

CytoSoft®platescanalsobeusedtoshowhowfibroblastsareabletodiscriminatebetweentheunderlyingstiffness.Thisismanifestedinbothadhesionsizeandstressfibers,asseenbelow.Itappearsthatthecellsonthe8kPastiffnesshavereducedintracellulartensionandincreasedadhesion.

ProductReferences

ReferencesforCytoSoft®Products:

1.Modaresi,Saman,etal.“DecipheringtheRoleofSubstrateStiffnessinEnhancingtheInternalizationEfficiencyofPlasmidDNAinStemCellsUsingLipid-BasedNanocarriers.”Nanoscale,vol.10,no.19,2018,pp.8947–8952.,doi:10.1039/c8nr01516c.

2.Wilson,ChristinaL.InVitroModelsOfBrainForStudyOfMolecularMechanismsInBrainDisorder.TheUniversityofNEBraska-Lincoln,2016.

3.Wilson,C.L.,Hayward,S.L.,&Kidambi,S.(2016).Astrogliosisinadish:Substratestiffnessinducesastrogliosisinprimaryratastrocytes.RSCAdvances,6(41),34447-34457.doi:10.1039/c5ra25916a

4.Prager-KhoutorskyM,LichtensteinA,KrishnanR,RajendranK,MayoA,KamZ,GeigerB,BershadskyAD.Fibroblastpolarizationisamatrix-rigidity-dependentprocesscontrolledbyfocaladhesionmechanosensing.Nat.CellBiol.2011;13:1457–1465.

5.GutierrezE,TkachenkoE,BesserA,SunddP,LeyK,DanuserG,GinsbergMH,GroismanA.HighRefractiveIndexSiliconeGelsforSimultaneousTotalInternalReflectionFluorescenceandTractionForceMicroscopyofAdherentCells.PLoSOne.2011;6:e23807.

6.MerkelR,KirchgessnerN,CesaCM,HoffmannB.Cellforcemicroscopyonelasticlayersoffinitethickness.Biophys.J.2007;93:3314–23.

7.Schellenberg,A.etal.Matrixelasticity,replicativesenescenceandDNAmethylationpatternsofmesenchymalstemcells.Biomaterials35,6351–8(2014).

8.Cesa,C.M.etal.Micropatternedsiliconeelastomersubstratesforhighresolutionanalysisofcellularforcepatterns.Rev.Sci.Instrum.78,034301(2007).

9.Gutierrez,E.&Groisman,A.MeasurementsofElasticModuliofSiliconeGelSubstratewithaMicrofluidicDevice.PlosOne6(2011).

10.Mori,H.,Takahashi,A.,Horimoto,A.,andHara,M.Migrationofglialcellsdifferentiatedfromneurosphere-formingneuralstem/Progenitorcellsdependsonthestiffnessofthechemicallycross-linkedcollagengelsubstrate.NeuroscienceLetters,Vol.555,October(2013)

11.Banerjee,I.,Carrion,K.,Serrano,R.,Dyo,J.,Sasik,R.,Lund,S.etal.Cyclicstretchofembryoniccardiomyocytesincreasesproliferation,growth,andexpressionwhilerepressingTgf-βsignaling.JMolCellCardiol.2015;79:133–144

12.Vertelov,G.etal.Rigidityofsiliconesubstratescontrolscellspreadingandstemcelldifferentiation.Sci.Rep.6,33411;doi:10.1038/srep33411(2016).

13.TkachenkoE,RawsonR,LaE,etal.RigidSubstrateInducesEsophagealSmoothMuscleHypertrophyandEoEFibroticGeneExpression.TheJournalofallergyandclinicalimmunology.2016;137(4):1270-1272.e1.doi:10.1016/j.jaci.2015.09.020.

14.Sao,K.etal.MyosinIIgovernsintracellularpressureandtractionbydistincttropomyosin-dependentmechanisms.MolecularBIOLOGyoftheCell30,1170–1181(2019).

15.Cooper,J.G.etal.SpinalCordInjuryResultsinChronicMechanicalStiffening.JournalofNeurotrauma(2019).doi:10.1089/neu.2019.6540

ProductCertificateofAnalysis

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SafetyandDocumentation

SafetyDataSheet

CertificateofOrigin

ProductDisclaimer

ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.

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