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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Department of Physiology and Biophysics, University of California, Irvine (UCI)
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Matheu, M. P., Cahalan, M. D. Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification. J. Vis. Exp. (9), e409, doi:10.3791/409 (2007).
从主要来源细胞的分离是必要的步骤，在许多更复杂的协议。美天旎提供的工具包，隔离从几个生物体的细胞，包括人类在内的非人类灵长类动物，老鼠，我们在这里描述，老鼠。基于磁珠细胞分离，使无论是正面的选择（或细胞耗竭）以及阴性选择。在这里，我们展示了不变或NA已经CD4 +辅助性T细胞的阴性选择。使用这个标准的协议，我们通常净化细胞≥96％纯的CD4 + / CD3 +。该协议是在与协议夹层和出版发行的朱庇特7，纯化的T细胞和其他类型的细胞过继转移成像目的，小鼠的外周淋巴结的双光子成像一起使用。虽然我们并没有表现出在这个协议的视频的流式细胞仪分析，它是强烈建议使用相应的抗体，通过流式细胞仪分离出的细胞检查的整体纯度。此外，我们证明T细胞隔离的非无菌的方法。如果无菌细胞是为您的特定最终用户的应用需要，一定要做好标准的无菌条件下，在组织培养罩中表现出的程序。感谢您看运气好，并用自己的实验！
请阅读并遵守协议http://www.miltenyibiotec.com/包括在美天旎套件或在网上。
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从主要来源细胞的分离是必要的步骤，在许多更复杂的协议。美天旎提供的工具包，隔离从几个生物体的细胞，包括人类在内的非人类灵长类动物，老鼠，我们在这里描述，老鼠。基于磁珠细胞分离，使无论是正面的选择（或细胞耗竭）以及阴性选择。在这里，我们展示了不变或NA已经CD4 +辅助性T细胞的阴性选择。使用这个标准的协议，我们通常净化细胞≥96％纯的CD4 + / CD3 +。虽然我们并没有表现出在这个协议的视频的流式细胞仪分析，它是强烈建议使用相应的抗体，通过流式细胞仪分离出的细胞检查的整体纯度。此外，我们证明T细胞隔离的非无菌的方法。如果无菌细胞是为您的特定最终用户的应用需要，一定要做好标准的无菌条件下，在组织培养罩中表现出的程序。
Subscription Required. Please recommend JoVE to your librarian.
我们要感谢编写的Mac缓冲丽贝卡Paquett。
| Name | Type | Company | Catalog Number | Comments |
| MACS Buffer | Reagent | None | None | Prepare as described by Miltenyi. |
| CD4+ T cell Isolation Kit: Untouched Isolation | Reagent | Miltenyi Biotec | 130-090-860 | For use with the Miltenyi system. |
| Sterile Phosphate-Buffered Saline (PBS) | Reagent | For use in the preparation of MACS buffer. | ||
| Corning 35mm Not TC-Treated Culture Dish | Other | Corning | 430588 | Used in the preparation of a single cell suspension. |
| 70 micron Cell Strainer | Tool | BD Biosciences | 352350 | Used in the preparation of a single cell suspension. |
| 6 mL Syringe | Tool | Tyco Healthcare, Covidien | 8881516051 | Used in the preparation of a single cell suspension. |
| 10x Dulbecco’s Phosphate Buffered Saline (DPBS) | Reagent | Invitrogen | 14200-075 | For use in RBC lysis. |
| Sterile Water | Reagent | Sigma-Aldrich | W4502 | For use in RBC lysis. |
1. Miller, M. J., Hejazi, A. S., Wei, S. H., Cahalan, M. D. & Parker, I. T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node. Proc Natl Acad Sci U S A 101, 998-1003 (2004).
2. Matheu, M. P., Deane, J. A., Parker, I., Fruman, D. A. & Cahalan, M. D. Class IA phosphoinositide 3-kinase modulates basal lymphocyte motility in the lymph node. J Immunol 179, 2261-9 (2007).
Comment: Typo in water lysis portion of the protocol. Font for microliters translated as mL instead of uL. Use 900 uL of ddH20 and 100 uL of 10x PBS.
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ReplyPosted by: Melanie M.August 13, 2008, 12:54 AM