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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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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原液分装。抗体孵育之前稀释。
做WB的浓度不一定需要很高的。可以设置简单梯度1:200；1:500；1:1000 。看结果再优化
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。
当然做都是可以做，需要带上阴性和阳性的对照组，以区别出是抗体制备的问题还是抗原决定簇被破坏的问题。
第一，血清成分复杂，最好用密理博的Montage Albumin Deplete Kit去除血清里50%的白蛋白
第二，磷酸化蛋白提取蛋白的时候，最好加入原钒酸钠，和磷酸酶抑制剂，防止蛋白脱磷酸化。
第三，不要用牛奶做封闭剂，用BSA。因为牛奶中含有磷酸化酪蛋白，防止非特异带。
第四，如果条带杂带多，而且，条带弱，可以选择millipore的signal boost，增加条带特异性，而且，增强条带的亮度。
原理简介：
本试剂盒采用改进SDS-碱裂解法裂解细胞，离心吸附柱内的硅基质膜在高盐，低pH值状态下选择性地结合溶液中的质粒DNA，再通过去蛋白液和漂洗液将杂质和其它细菌成分去除，最后低盐，高pH值的洗脱缓冲液将纯净质粒DNA从硅基质膜上洗脱。
注意事项：
◆ 第一次使用时，将试剂盒所带全部的RNase A加入溶液P1后（终浓度100ug/ml）置于4℃保存。如果溶液P1中RNase A失活，提取的质粒可能会有混杂有微量RNA残留， 这时可在溶液P1中补加RNase A即可。
◆ 第一次使用前请先在15ml漂洗液WB中加入45ml无水乙醇，加入后请及时在方框打钩标记已加入乙醇，以免多次加入!
◆ 温度低时溶液P2中SDS可能会出现浑浊或者析出沉淀，可在37℃水浴加热几分钟，即可恢复澄清，不要剧烈摇晃，以免形成过量的泡沫。
◆ 避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化，各溶液使用后应及时盖紧盖子。
试剂盒特点：
◆ 产量高---一次提取高达30ug以上的质粒。
◆ 纯度高---OD260/OD280一般为1.80~1.85本试剂盒提取的质粒纯度好，能充分保证测序所需要的读长(用于ABI3730测序一般可达1000bp有效读长)。
◆ 快速，方便，不需要使用有毒的苯酚，氯仿等试剂，也不需要乙醇沉淀。
提示
BIOTEKE的质粒提取试剂盒既适用于革兰氏阴性菌中质粒的提取，同时也可从革兰氏阳性菌中提取质粒。由于革兰氏阳性菌外被一层较厚的细胞壁，会严重阻碍细菌细胞的裂解，因此必须在裂解细胞前破除，方法如下：
收集适量的菌体，加入250ul溶液P2，充分悬浮菌液，加入溶菌酶使其终浓度在10-20mg/ml左右在37℃处理30分钟左右。加入溶菌酶的浓度和处理的时间可根据不同的菌主和具体实验条件进行调整。
