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,andMethod | CytoSoft®DiscoveryKit,6-wellPlates#5190-7EA |
SterilizationMethod | Ozone |
PlateSize | 6-WellPlates |
QuantityperPackage | 7Plates |
Rigidty(ElasticModulus) | 0.2,0.5,2,8,16,32,64kPa(exactvaluesprovidedontheCofA) |
Storage | RoomTemperature |
ShelfLife | Minimumof6monthsfromdateofreceipt |
PlateSurfaceMaterial | FunctionalizedSilicone |
GrowthAreaperWell | 9.5cm2 |
TypicalWorkingVolumeperWell | 2.0to3.5mL |
CellAttachmentAssay | Pass |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
CoatingProcedure
Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.
RemovetheCytoSoft®productfromtheprotectivesleeveinasterilehood.
- Prepareextracellularmatrixmaterialbyneutralizinginamine-freebufferpH7.4to7.9(suchas1XDPBS).WedonotrecommendusinggelatinasyourECMprotein.
Note:Pre-warmthecoatingsolutiontoapproximatelyroomtemperaturebeforeuse.
- Diluteasneeded,anddispense3mlofsolutionintoeachwelltocoatthesurface.
Note:RecommendeddilutionforPureCol®TypeIcollagenis1:30(~100µg/ml).
Note:Thehydrophobicsurfacerequireslargervolumestocoverthesurfacethandoconventionalplasticdishes
- IncubateECMcoatedCytoSoft®atroomtemperature,coveredfor0.5-1hour.
- 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
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SafetyDataSheet
CertificateofOrigin
ProductDisclaimer
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
美国AdvancedBioMatrix(简称ABM) www.advancedbiomatrix.comAdvancedBioMatrix(简称ABM)是美国一家著名的生物公司,获得了AllerganInc的授权(Allergan用25年时间不断完善胶原蛋白相关的产品的生产工艺),将Allergan的专业和技术用于蛋白生产与检测,致力于为组织工程、细胞分析及细胞增殖等研究领域提供优质稳定的产品。AdvancedBioMatrix不断丰富已有产品线,目前可为三维细胞培养提供各种胶原蛋白、纤连蛋白、玻连蛋白、水性凝胶、不同粘度与分子量的透明质酸以及低代成纤维细胞等。在美国全部产品授权Sigma销售。AdvancedBioMatrix是组织培养,细胞分析和细胞增殖三维(3D)应用的生命科学领域的领导者。我们的产品被公认为纯度,功能性和一致性的标准。我们在生产,分离,纯化,冷冻干燥,细胞培养和蛋白质测试,粘附肽,附着因子,底物刚性和其他3D矩阵产品方面拥有丰富的专业知识。我们的专业技术和知识正在被用来确保我们的产品质量最高,批次之间一致且易于为我们的研究客户使用。
美国AdvancedBioMatrix是3D组织培养、细胞检测和细胞增殖等领域实验解决方案的佼佼者。AdvancedBioMatrix在分离、纯化、冻干、细胞培养和蛋白检测、多肽粘附、附着因子、基质硬度和其他3Dmatrix 产品开发方面有着丰富的经验。AdvancedBioMatrix的研发经验和专业知识确保其产品可达到最佳质量,并保证产品之间一致性,方便研究客户使用。以下为AdvancedBioMatrix3DMatrices 产品竞争优势:1. 提供高纯度和成分确定的胞外基质;2. 超过1000余篇文献引用PureCol产品,品质非常均一;3. 在3D培养基领域可提供最全面的产品线;4. 唯一可提供特异性刚性有机硅基板的公司(CytoSoft);5. 唯一可提供可溶性丝纤蛋白的供应商(可运用于多种3D培养);6. 如果客户首次接触3D胶原凝胶,AdvancedBioMatrix还是唯一的预制胶原蛋白(PureColEZGel)供应商;
以下产品为AdvancedBioMatrix全球畅销品:1.PureCol 牛源I型胶原蛋白 3mg/ml#5005-100ML2.Nutragen牛源I型胶原蛋白 6mg/ml#5010-50ML3.FibriCol 牛源I型胶原蛋白 10mg/ml#5133-20ML4.VitroCol 人源I型胶原蛋白 #5007-20ML5. 弹性蛋白原 #5052-1MG6.ECMSelectArraykitUltra-36#5170-1EA7.CytoSoft(刚性可变的基底,AdvancedBioMatrix最新添加产品5190-7EA)8. 人III型胶原蛋白 #5021-10MG9. 人IV型胶原蛋白 #5022-5MG10.SilkFibroin溶液 #5154-20ML11.Fibronectin#5080-5MG12.Vitronectin#5051-0.1MG
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1、如果是提取的总蛋白,然后做WB,用β-actin或者GAPDH做内参肯定是没有问题的,这是公认的东西。
2、如果用膜蛋白提取试剂盒提取蛋白,再用β-actin作为内参似乎不妥,因为理论上来讲β-actin在膜上是不表达的。WB能做出β-actin来是因为膜蛋白提取时把胞质蛋白也提出来了。然而,如果我们试验目的是用药物处理细胞,比较处理前后某种膜蛋白的表达情况,此时用膜蛋白提取试剂盒提取膜蛋白后再用β-actin做内参似乎就不妥了,因为你根本不知道药物处理前后混杂了多少的胞质蛋白进来。如果没有胞质蛋白混进去的话,β-actin就是检测不到的。
转膜后就要区分与胶接触的一面与另一面了。话说楼主好奇的话可以跑蛋白的试试看哈。比如目的蛋白的胶用光滑面贴着胶,内参蛋白用粗糙面贴着胶一起转膜试试转膜结果看看呢。
考马斯亮兰染液也可以重复用。新配的染液10分钟即可,重复3次后要染30分钟。
一抗二抗可以重复利用,但是注意要在5%milk中加入0.2% sodium azide ,并且用完以后放入4度保存,我的经验重复使用4-5次是肯定没有问题的.如果保存不当,就会污染微生物,只能丢弃.
取名尿素。尿素含氮46%,是固体氮肥中含氮量最高的。尿素在酸、碱、酶作用下(酸、碱需加热)能水解生成氨和二氧化碳。希望我能帮助你解疑释惑。
之前是担心所需组织量不够(要求是200-300mg,我的组织只有20mg左右)才跑不出来,后来换用大鼠心脏测试,组织量绝对够。。。而且在跑western之前进行了说明书上的蛋白浓缩步骤,可是在加loADIngbuffer之后,蛋白沉淀怎么都溶解不掉~说明书也注明了如果有沉淀可以取上清继续上样,可是每次上样感觉样品往上浮,只有部分样品往孔里沉,而且跑出来什么都没有,连内参都没有。用的是β-actin的内参。。
western之前浓缩步骤如下:
1、取所得提取物,每100ul膜蛋白加入约300ul的溶解buffer和约100ul三氯乙酸(TCA)试剂,混匀后置冰上20-30min后,13000rpm,离心15min,尽可能去除上清。
2、沉淀加入1ml丙酮,室温静置10min后,13000rpm离心15min。
3、弃上清,沉淀真空旋干或置冰上干燥10min(敞开离心管盖),按适当体积比加入loadingbuffer(使用前没100ulloadingbuffer加入2-5ulβ-巯基乙醇)溶解,彻底分散(枪头反复吹吸或剧烈涡旋),煮沸5min。【注:加入loadingbuffer后如有部分难容物,可取上清继续上样。】