Product Description
The Ruthenium visible light photocrosslinking kit is ideal for tissue engineering, cell culture, and bioprinting, where tuning the mechanical properties of the substrate is required. The kit provides enough photoinitiator for >200 mL of bioinks/hydrogels (following recommended concentrations).
- Ruthenium is a photoinitiator that utilizes visible light photocrosslinking (400-450nm) to covalently crosslink free tyrosine and acryl groups.
- Ruthenium photoinitiator has been tested on collagen type I, gelatin, silk fibroin, methacrylated hyaluronic acid, methacrylated gelatin, methacrylated collagen type I and PEGDA.
- Ruthenium is water soluble and yields better cell cytocompatibility, and crosslinking efficiency.
- Increasing Visible light intensity from 3-100 mW/cm2 using Ruthenium did not significantly decrease cell viability from 90%.
- Increasing Ruthenium concentration by 10 times (10x) did not decrease cell viability from 90%.
- Ruthenium is red/yellow/orange in color and will change the color of your solutions, hydrogels, or printed constructs.
This Ruthenium photocrosslinking kit is considered non-sterile. Adding antibiotics to your cell culture system, or sterile filtering is recommended. To sterile filter, resuspend the entire volume of Ruthenium and Sodium Persulfate (separately) and filter through small 0.2 micron button filters (separately). Use the sterile photoinitiator within 2 weeks.
The Ruthenium visible light photocrosslinking kit is composed of two components, as found in Table 1.
Table 1:
| Item | Catalog Number | Package Size |
| Ruthenium Photoinitiator | #5246 | 200 mg |
| Sodium Persulfate Photoinitiator | #5247 | 1 gram |
Storage/Stability:
The product ships ambient. Store the kit at room temperature. Weigh out the required amount of powder to solubilize. Once solubilized, use the Ruthenium and Sodium Persulfate within 2 weeks.
Dry powder (non-solubilized) is stable for >1 year at room temperature.
Directions for Use
Download the full PDF versionor continue reading below:
1. Calculate desired volume of hydrogel or bioink (ECM + cells).
2. Multiply desired volume by 0.02. This is how much Ruthenium and Sodium Persulfate (each) you will be adding to your pre-hydrogel solution.
3. Solubilize required Ruthenium in water or 1X PBS at a concentration of 37.4 mg/mL.
4. Solubilize Sodium Persulfate in water or 1X PBS at a concentration of 119 mg/mL.
5. Add the calculated volume (step 2) of Ruthenium to your pre-hydrogel solution and thoroughly mix.
6. Add the calculated volume (step 2) of Sodium Persulfate to your pre-hyrogel solution and thoroughly mix.
Notes:Do not mix the Ruthenium and Sodium Persulfate together prior to adding to the pre-hydrogel. You will get a rapid redox reaction and they will precipitate instantaneously.
7. Add in cells, if desired.
8. Photocrosslink at 400-450nm wavelength. Initial recommendation is 50 mW/cm2 for >3 minutes to maintain shape/hydrogel fidelity. You may tune photoinitiator concentration, light intensity and Photocrosslinking time to customize final hydrogel stiffness.
Additional Notes:
If using neutralized type I collagen, you can allow the collagen to polymerize at 37C to form a hydrogel, and then photocrosslink to further crosslink and modulate the gel stiffness.
How to use Ruthenium with Lifeink® 200 and 3D Bioprinting:
1. Print Lifeink® 200 according to the recommended protocols, into the FRESH support slurry.
2. After printing, incubate the print to melt the FRESH gelatin support slurry.
3. After ~30 minutes, replace the melted gelatin with warm cell culture media.
4. For example, pipette out 2 mL of melted gelatin, and then add in 2 mL of media. Repeat until the gelatin is removed.
5. Prepare stock solutions of Ruthenium and Sodium Persulfate (found in the above protocol).
6. Multiply total volume of media that your Lifeink® 200 structure is floating in. Multiply by 2%, and add that amount of Ruthenium and Sodium Persulfate to the cell culture media (directly in the same dish that your structure is in).
7. Photocrosslink with visible light (400-450 nm) until desired crosslinking is achieved.
8. Gently replace the media with fresh media, as done in step 3.
Product Q & A
The full abosrbance spectrum can be found here:https://iopscience.iop.org/1758-5090/10/3/034101/media/BFaac00c_suppdata.pdf
Product Cell Assay
Cells tested within 3D hydrogels crosslinked with Ruthenium and visible light photocrosslinking:
Product References
References for Ruthenium:
1. Parrish, J., Lim, K. S., Baer, K., Hooper, G. J., & Woodfield, T. B. F. (2018). A 96-well microplate bioreactor platform supporting individual dual perfusion and high-throughput assessment of simple or biofabricated 3D tissue models.Lab on a Chip. Advance online publication.doi: 10.1039/c8lc00485d
2. Parker, J. D., Lim, K. S., Kieser, D. C., Woodfield, T. B. F., & Hooper, G. J. (2018). Is tranexamic acid toxic to articular cartilage when administered topically? What is the safe dose?Bone & Joint Journal,100-B(3), 404-412.doi: 10.1302/0301-620X.100B3.BJJ-2017-1135.R1
3. Lim, K. S., Levato, R., Costa, P. F., Castilho, M. D., Alcala-Orozco, C. R., van Dorenmalen, K. M. A., … Hooper, G. J., … Woodfield, T. B. F. (2018). Bio-resin for high resolution lithography-based biofabrication of complex cell laden constructs.Biofabrication,10, 034101.doi: 10.1088/1758-5090/aac00c
4. Bertlein, S., Brown, G., Lim, K. S., Jungst, T., Boeck, T., Blunk, T., … Hooper, G. J., Woodfield, T. B. F., & Groll, J. (2017). Thiol-ene clickable gelatin: A platform bioink for multiple 3D biofabrication technologies.Advanced Materials,29(44), 1703404.doi: 10.1002/adma.201703404
5. Mekhileri, N. V., Lim, K. S., Brown, G. C. J., Mutreja, I., Schon, B. S., Hooper, G. J., & Woodfield, T. B. F. (2017). Automated 3D bioassembly of micro-tissues for biofabrication of hybrid tissue engineered constructs.Biofabrication. Advance online publication.doi: 10.1088/1758-5090/aa9ef1
6. Lim, Khoon S., et al. "New visible-light photoinitiating system for improved print fidelity in gelatin-based bioinks."ACS Biomaterials Science & Engineering2.10 (2016): 1752-1762.
7. Lim, K. S., Ramaswamy, Y., Roberts, J. J., Alves, M.-H., Poole-Warren, L. A., & Martens, P. J. (2015). Promoting cell survival and proliferation in degradable poly(vinyl alcohol)-tyramine hydrogels.Macromolecular Bioscience,15(10), 1423-1432.doi: 10.1002/mabi.201500121
8. Lim, K. S., Alves, M. H., Poole-Warren, L. A., & Martens, P. J. (2013). Covalent incorporation of non-chemically modified gelatin into degradable PVA-tyramine hydrogels.Biomaterials,34(29), 7097-7105.doi: 10.1016/j.biomaterials.2013.06.005
9. Green, R. A., Lim, K. S., Henderson, W. C., Hassarati, R. T., Martens, P. J., Lovell, N. H., & Poole-Warren, L. A. (2013). Living electrodes: Tissue engineering in the neural interface.Proceedings of the 35th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBS).(pp. 6957-6960). IEEE.doi: 10.1109/EMBC.2013.6611158
Product Certificate of Analysis
Safety and Documentation
Safety Data Sheet
Product Disclaimer
This 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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两个意义不一样啊，定性是确定你抗体是阴还是阳的，如果阳证明2个情况，一个是你感染了丙肝病毒，另一个是你曾经感染过这个病毒，现在已经好了。但是到底是哪个情况还要进一步做定量测试既病毒RNA检测。如果这个测试值在最低线以下就证明你现在没事，不具有传染性也不是患者只是携带者；如果高于最低线那你就是患者了，就需要治疗
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
如果要是直接注射，就不知道了
加入荧光标记探针，巧妙地把核算扩增、杂交、光谱分析和实时检测技术结合在一起，借助于荧光信号来检测PCR产物。一方面提高了灵敏度，另一方面还可以做到PCR每循环一次就收集一个数据，建立实时扩增曲线，准确地确定CT值，从而根据CT值确定起始DNA的拷贝数，做到真正意义上的DNA定量。另外由于CT值是一个完全客观的参数，CT值越小，模版DNA的起始拷贝数越小。因此，利用CT值确定DNA拷贝数实时PCR方法比普通终点定量方法更加准确
试剂盒里有详细的说明书，告诉你样品需要多少量，每个试剂需要加入多少量，和详细的实验步骤，一般买来就可以用，不用人教。
所以你问一个样需要多少量是没法回答的，测定过程是要加很多种试剂的。
