Product DescriptionThisCytoSoft®6-well plate product has a defined elastic modulus (see certificate of analysis) in a standard flat-bottom 6-well plate. The thickness of the silicone gel is uniform with a ~0.5 mm thick layer of silicone in each well that is fully compatible with mammalian cell cultures. The silicone gels are activated and ready to bind to a purified ECM, such as PureCol® type I collagen (#5005) prior to cell addition. The plates are packaged individually and sterilized, and provided with 5 plates per package.The rigidity of the substrate to which cells adhere can have a profound effect on cell morphology and gene expression.CytoSoft®products provide a tool to culture cells on substrates with various rigidities covering a broad physiological range. On the bottom of each well, there is a thin layer of specially formulated biocompatible silicone, whose elastic modulus (rigidity) is carefully measured and certified. The surfaces of the gels inCytoSoft®products are functionalized to form covalent bonds with amines on proteins. This chemical functionalization is stable and the reaction does not require a catalyst, facilitating the coating of the gel surfaces with matrix proteins and plating cells.The silicone substrates ofCytoSoft®products are optically clear and have a low auto-florescence. The layer of silicone in each well is firmly bonded to the bottom of the well. Unlike hydrogels (such as polyacrylamide gels), silicone gels are not susceptible to hydrolysis, do not dry nor swell, are resilient and resistant to tearing or cracking, and their elastic moduli (rigidities) remain nearly unchanged during extended storage periods.CytoSoft®products accommodate the harvesting of cells using enzymes such as trypsin and collagenase. There is no biochemical breakdown of the substrate during or after enzyme treatment, and there are no residuals of the substrate in the sample retrieved from aCytoSoft®plate.Parameter, Testing, and MethodCytoSoft®6-Well PlatesSterilization MethodOzonePlateSize6-Well PlatesQuantity per Package5PlatesRigidty (Elastic Modulus)0.2, 0.5, 2, 8, 16, 32, 64 kPa (exact values provided on the CofA)Storage TemperatureRoom TemperatureShelf LifeMinimum of 6 months from date of receiptPlate Surface MaterialFunctionalized SiliconeGrowth Area per Well9.5 cm2Typical Working Volume per Well2.0 to 3.5 mLCell Attachment AssayPassDirections for UseDownload the full PDF versionor continue reading below:Coating ProcedureNote:Use these recommendations as guidelines to determine the optimal coating conditions for your culture system.Remove theCytoSoft®product from the protective sleeve in a sterile hood.Prepare extracellular matrix material by neutralizing in amine-free buffer pH 7.4 to 7.9 (such as 1X DPBS). We do not recommend using gelatin as your ECM protein.Note: Pre-warm the coating solution to approximately room temperature before use.Dilute as needed, and dispense 3 ml of solution into each well to coat the surface.Note:Recommended dilution for PureCol®Type I collagen is 1:30 (~100 µg/ml).Note: The hydrophobic surface requires larger volumes to cover the surface than do conventional plastic dishesIncubate ECM coatedCytoSoft®at room temperature, covered for 0.5-1 hour.After incubation, aspirate any remaining material and rinse coated surfaces immediately two times with culture medium or PBS. Leave about 2.5 ml of medium per well to keep surface covered.Note: Do not allow theCytoSoft® surface to become dry once the surface has been wetted.5. Coated surfaces are ready for use.Standard harvesting procedures used for removing cells from cultureware can be employed for harvesting cells from theCytoSoft®product including use of trypsin, Accutase® and non-enzymatic cell detachment solutions.Product Q & AWhat objective is recommended for CytoSoft® Imaging Plates?We recommend a 60X objective for the imaging plates.How do you measure the stiffness of the CytoSoft® plates?The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels.How do I avoid the formation of bubbles on the PDMS surface?Using prewarmed media will decrease gas solubility and help prevent bubbles on the surface of the silicone.Why should the CytoSoft® surfaces not be allowed to dry?The silicone gel can crack if the surface becomes dry.Are the CytoSoft® plates reusable?No. Cell matrix proteins are attached to the surface of the gel via covalent bonding. It is difficult to form a new ECM layer after cell detachment. There are no longer any reactive groups on the surface of the gel after the initial cell culture.Is DIC possible with CytoSoft®?The change in the refractive index of the silicone distorts things a little, so DIC will be possible but not perfect. Also, DIC is only possible on the glass bottom imaging plates, not the 6-well plastic plates.What is the refractive index of CytoSoft®?1.41My cells are not growing well - what could the problem be?The two main issues are:1. Ineffecient coating of the matrix proteins due to low pH of the coating solution.2. Insufficient wash of leftover cell matrix after the coating procedure.Can CytoSoft® be coated with Hyaluronic Acid or other Non-ECM proteins?No. Cytosoft must be coated with an ECM protein such as Collagen or Fibronectin.What functional groups are active on the PDMS surface?The surface is decorated with anhydride functional groups.Will the mechanical properties of the PDMS change if exposed to plasma?Plasma use is not recommended. Plasma will induce formation of a hard crust on the surface and will change the mechanical properties of the CytoSoft® products.Is it OK if I accidentally freeze CytoSoft®?Do not freeze the CytoSoft® products.When frozen, there is a good chance that the silicone surface gets hydrolyzed and absorbs moisture, which would inactivate the binding sites and make the product not-usable.If you froze the product and it is still frozen, warm the CytoSoft® up at 60C with the bag vented. That will minimize the chance of them absorbing moisture – but there is still a chance that they will no longer be functional.Can I coat CytoSoft® with Gelatin?Using gelatin for coating CytoSoft is often problematic because gelatin often has low molecular weight impurities that block binding sites on the activated surface of the silicone. We recommend using highly purified ECM's instead. Product Cell AssayCytoSoft®plates can also be used to show howfibroblastsare able to discriminate between the underlying stiffness. This ismanifested in both adhesion size andstress fibers, as seen below. It appears that the cells on the 8 kPa stiffness havereduced intracellular tension and increasedadhesion.Product ReferencesReferences for CytoSoft® Products:1. Modaresi, Saman, et al. “Deciphering the Role of Substrate Stiffness in Enhancing the Internalization Efficiency of Plasmid DNA in Stem Cells Using Lipid-Based Nanocarriers.”Nanoscale, vol. 10, no. 19, 2018, pp. 8947–8952., doi:10.1039/c8nr01516c.2. Wilson, Christina L.In Vitro Models Of Brain For Study Of Molecular Mechanisms In Brain Disorder. The University of Nebraska-Lincoln, 2016.3. Wilson, C. L., Hayward, S. L., & Kidambi, S. (2016). Astrogliosis in a dish: Substrate stiffness induces astrogliosis in primary rat astrocytes.RSC Advances,6(41), 34447-34457. doi:10.1039/c5ra25916a4. Prager-Khoutorsky M, Lichtenstein A, Krishnan R, Rajendran K, Mayo A, Kam Z, Geiger B, Bershadsky AD. Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing. Nat. Cell Biol. 2011; 13:1457–1465.5. Gutierrez E, Tkachenko E, Besser A, Sundd P, Ley K, Danuser G, Ginsberg MH, Groisman A. High Refractive Index Silicone Gels for Simultaneous Total Internal Reflection Fluorescence and Traction Force Microscopy of Adherent Cells. PLoS One. 2011; 6:e23807.6. Merkel R, Kirchgessner N, Cesa CM, Hoffmann B. Cell force microscopy on elastic layers of finite thickness. Biophys. J. 2007; 93:3314–23.7. Schellenberg, A. et al. Matrix elasticity, replicative senescence and DNA methylation patterns of mesenchymal stem cells. Biomaterials 35, 6351–8 (2014).8. Cesa, C. M. et al. Micropatterned silicone elastomer substrates for high resolution analysis of cellular force patterns. Rev. Sci. Instrum. 78, 034301 (2007).9. Gutierrez, E. & Groisman, A. Measurements of Elastic Moduli of Silicone Gel Substrate with a Micro fluidic Device. Plos One 6 (2011).10. Mori, H., Takahashi, A., Horimoto, A., and Hara, M. Migration of glial cells differentiated from neurosphere-forming neural stem/progenitor cells depends on the stiffness of the chemically cross-linked collagen gel substrate. Neuroscience Letters, Vol. 555, October (2013)11. Banerjee, I., Carrion, K., Serrano, R., Dyo, J., Sasik, R., Lund, S. et al.Cyclic stretch of embryonic cardiomyocytes increases proliferation, growth, and expression while repressing Tgf-β signaling.J Mol Cell Cardiol.2015;79:133–14412. Vertelov, G.et al. Rigidity of silicone substrates controls cell spreading and stem cell differentiation.Sci. Rep.6, 33411; doi: 10.1038/srep33411 (2016).13. Tkachenko E, Rawson R, La E, et al. Rigid Substrate Induces Esophageal Smooth Muscle Hypertrophy and EoE Fibrotic Gene Expression.The Journal of allergy and clinical immunology. 2016;137(4):1270-1272.e1. doi:10.1016/j.jaci.2015.09.020.14. Sao, K.et al.Myosin II governs intracellular pressure and traction by distinct tropomyosin-dependent mechanisms.Molecular Biology of the Cell30,1170–1181 (2019).15.Cooper, J. G.et al.Spinal Cord Injury Results in Chronic Mechanical Stiffening.Journal of Neurotrauma(2019). doi:10.1089/neu.2019.6540Product Certificate of AnalysisNo result for .Product VideosWhat is CytoSoft®?VideoSee More >How to Coat CytoSoft® PlatesVideoSee More > See More Safety and DocumentationSafety Data SheetCertificate of OriginProduct DisclaimerThis product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.

美国Advanced BioMatrix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。Advanced BioMatrix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3D matrix 产品开发方面有着丰富的经验。Advanced BioMatrix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Advanced BioMatrix 3D Matrices 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，Advanced BioMatrix还是唯一的预制胶原蛋白(PureCol EZ Gel)供应商。

以下产品为Advanced BioMatrix全球畅销品：
1. PureCol 牛源I型胶原蛋白 3 mg/ml #5005-100ML
2. Nutragen牛源I型胶原蛋白 6 mg/ml #5005-100ML
3. FibriCol 牛源I型胶原蛋白 10 mg/ml #5133-20ML
4. VitroCol 人源I型胶原蛋白 #5007-20ML
5. 弹性蛋白原 #5052-1MG
6. ECM Select Array kit Ultra-36 #5170-1EA
7. CytoSoft（刚性可变的基底，Advanced BioMatrix最新添加产品5190-7EA）
8. 人III型胶原蛋白 #5021-10MG
9. 人IV型胶原蛋白 #5022-5MG
10. Silk Fibroin溶液 #5154-20ML
11. Fibronectin #5080-5MG
12. Vitronectin #5051-0.1MG

Advanced BioMatrix最畅销的产品是PureCol（5005-100ML）。目前已有上千发表的论文使用这款产品。（Nutragen 5010、fibricol 5133和PureCol的物质成分一样，仅仅是浓度不同）浓度越高，胶原蛋白硬度越强。