The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.
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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.
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School of Engineering, University of California Merced - UC Merced
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Chen, C., Pegan, J., Luna, J., Xia, B., McCloskey, K., Chin, W., et al. Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies. J. Vis. Exp. (13), e692, doi:10.3791/692 (2008).
胚体（EB）的胚胎干细胞的聚集。创建这些聚集的最常用的方法是悬滴法，孔板移液到任意数量的细胞费力的方法。被迫彼此接近的干细胞之间的相互作用，促进了EBS的一代。因为在每个井的媒体每天要手动交换，这种做法是手动密集。
此外，因为环境参数，包括细胞，细胞可溶性因子的相互作用，pH值，和氧气供应的EB大小的功能，从传统的挂滴获得的细胞群可以相差很大，即使在相同条件下培养。的确，最近的研究表明，初步形成总额的细胞数量有重大影响干细胞分化。我们已经开发出一种简单，快速，可伸缩的文化的方法加载到预先定义的数字，微型井细胞胚体的发展和维护。最后，这些细胞是方便作进一步的分析和实验。这种方法适合任何实验室，无需专用设备。我们通过这种方法使用红色荧光的小鼠细胞株（129S6B6 F1），胚体。
1。制作Shrinky，丁克模具
2。使PDMS的微孔
3。补漏细胞在微孔
4。细胞孵育
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我们已经开发出一种简单，快速，可伸缩的培养方法加载到微井（Shrinky - Dinks成型）预先定义的细胞数目和他们保持胚体的发展。最后，这些细胞是方便作进一步的分析和实验。这种方法适合任何实验室，不需要专用的设备，因为我们排除光刻需要。我们可以改变的微孔的大小以及细胞/井的浓度改变胚体的数量和大小。
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我们要感谢支持这项工作摆围。安德拉什纳吉博士在西奈山医院，多伦多，安大略省的细胞系的慷慨捐赠。
| Name | Type | Company | Catalog Number | Comments |
| Shrink-Dink Film | Material | K&B Innovations | D300-10A | |
| PDMS | Material | Dow Corning | Sylgard 184 | |
| Acetone | Reagent | Fisher Scientific | A16P-4 | |
| Ethanol | Reagent | Fisher Scientific | A405P-4 | |
| PBS | Reagent | Sigma-Aldrich | P4417 | |
| BMP-4 | Reagent | R&D Systems | 314-BP-010 | |
| Knock-Out DMEM (KO DMEM) | Reagent | Invitrogen | 10829 | |
| KnockOut Sirum Replacement (KSR) | Reagent | Invitrogen | 10828 | |
| Penn-Strep | Reagent | Invitrogen | 15070-063 | |
| L-glutamine | Reagent | Invitrogen | 25030-081 | |
| Non-essential Amino Acids (NEAA) | Reagent | Invitrogen | 11140 | |
| D-mercaptoethanol (BME) | Reagent | Calbiochem | 444203 | |
| Leukemia Inhibitory Factor (LIF) {ESGRO 106 units} | Reagent | Chemicon International | ESG1106 | |
| Printer | Tool | Hewlett-Packard | Laser Jet 2420d | |
| Oven | Tool | Yamato Scientific | DP-22 | |
| For the mESC media(McCloskey lab protocol):(for total media prepared: 50ml; 100ml) KO DMEM: 40.8ml; 81.6ml15% KSR: 7.5ml; 15ml 1x Penn-Strep: .5ml; 1ml2mM L-glutamine: .5ml; 1mlNEAA : .5ml; 1mlLIF: 100ul; 200ulBMP-4 (10ng/ml): 50ul; 100ulDiluted BME: 50ul; 100ul(Add 35ul of sterile filtered BME to 5ml of PBS and syringe filter sterilize. Discard after 2 weeks. Final concentration in the solution is .1mM) | ||||
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What a useful video! Thank you very much for innovating this method and for making it so user-friendly for others in the scientific community.
Best,
Kriti Sharma
Duke University Dep't of Biology, Durham, NC
1
ReplyPosted by: KritiMay 1, 2008, 1:25 PM