ProductDescription
TheCytoSoft®-Imaging24-wellproductshaveadefinedelasticmodulus(seecertificateofanalysis)witha#1.5glassbottomattahcedtoa24-wellplate.Thethicknessofthesiliconegelisuniformwitha~0.03mmthicklayerofsiliconeineachwell.ThesiliconegelsareactivatedandreadytobindtoapurifiedECM,suchasPureCol®typeIcollagen(#5005)priortocelladdition.
Therigidityofthesubstratetowhichcellsadherecanhaveaprofoundeffectoncellmorphologyandgeneexpression.CytoSoft®productsprovideatooltoculturecellsonsubstrateswithvariousrigiditiescoveringabroadphysiologicalrange.
TheCytoSoft®–Imagingplateisforhigh-resolutionimagingwherelowautofluorescenceandexceptionalopticalclarityarerequired.Plateconsistsofa175µmthinpolycarbonatefilm-bottomplatebondedwithablackpolystyreneframeandincludesalid.Theplatesaresterilizedusingozoneandprovidedwith1plateperpackage.
Onthebottomofeachwell,thereisathinlayerofspeciallyformulatedbiocompatIBLesilicone,whoseelasticmodulus(rigidity)iscarefullymeasuredandcertified.ThesurfacesofthegelsinCytoSoft®productsarefunctionalizedtoformcovalentbondswithaminesonproteins.Thechemicalfunctionalizationisstableandthereactiondoesnotrequireacatalyst,facilitatingcoatingofthegelsurfaceswithmatrixproteinsandplatingcells.
Thesiliconesubstratesareopticallyclearandhaveanearzeroauto-florescence.Thelayerofsiliconeineachwellisfirmlybondedtothebottomofthewell.Unlikehydrogels(suchaspolyacrylamidegels),siliconegelsarenotsusceptibletohydrolysis,donotdryorswell,areresilientandresistanttotearingorcracking,andtheirelasticmoduli(rigidities)remainnearlyunchangedduringextendedstoragetimes.
CytoSoft®-Imagingproductsaccommodatelivecellstainingusingmembraneandcellpermeabledyes;fixationofcellsusingcommontechniquessuchasparaformaldexyde;andimmunostainingoffixedcells.
| Parameter,Testing,andMethod | CytoSoft®Imaging24-WellPlates |
| SterilizationMethod | Ozone |
| PlateSize | 24-MultiwellBlackPolystyrenePlatewithGlassBottom |
| QuantityperPackage | 1Plate |
| Rigidty(ElasticModulus) | 0.2,0.5,2,8,16,32,64kPa(exactvaluesprovidedontheCofA) |
| StorageTemperature | RoomTemperature |
| ShelfLife | Minimumof6monthsfromdateofreceipt |
| PlateSurfaceMaterial | FunctionalizedSilicone |
GrowthAreaperWell | 1.9cm2 |
TypicalWorkingVolumeperWell | 0.7to1.2mL |
CellAttachmentAssay | Pass |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
CoatingProcedure
Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.
CautionaryNote:Thebottomofa24-wellplatecanbedetachedbyexcessivemechanicalforcesuchascentrifugationordirectcontactwithliquidhandlingtools(tips,Pipettes,etc.).
RemovetheCytoSoft®productfromtheprotectivesleeveinasterilehood.
- Prepareextracellularmatrixmaterialbyneutralizinginamine-freebufferpH7.4to7.9(suchas1XDPBS).WedonotrecommendusinggelatinasyourECMprotein.
Note:Pre-warmthecoatingsolutiontoapproximatelyroomtemperaturebeforeuse.
- Diluteasneeded,anddispense1mlofsolutionintoeachwelltocoatthesurface.
Note:RecommendeddilutionforPureCol®TypeIcollagenis1:30(~100µg/ml).
Note:Thehydrophobicsurfacerequireslargervolumestocoverthesurfacethandoconventionalplasticdishes
- IncubateECMcoatedCytoSoft®atroomtemperature,coveredfor0.5-1hour.
- Afterincubation,aspirateanyremainingmaterialandrinsecoatedsurfacesimmediatelytwotimeswithculturemediumorPBS.Leaveabout2.5mlofmediumperwelltokeepsurfacecovered.
Note:DonotallowtheCytoSoft®surfacetobecomedryoncethesurfacehasbeenwetted.
- 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
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ProductDisclaimer
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
