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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Beeton, C., Chandy, K. G. Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies. J. Vis. Exp. (8), e326, doi:10.3791/326 (2007).
自然杀伤（NK）细胞的大颗粒属于先天免疫系统发挥重要的角色，在对抗癌症和感染的细胞毒性淋巴细胞，但也牵连在怀孕和移植排斥反应的早期阶段。这些细胞是外周血中，从它们可以被隔离。细胞可以分离出积极或消极的选择。我们正选择使用表面标记的抗体只对目前的利益，而细胞阴性选择感兴趣的所有细胞，但细胞中存在，我们使用有针对性的表面标记抗体鸡尾酒。后者的技术，提出了离开的抗体的利息细胞的优势，从而减少了不需要的细胞的活化或differenciation的风险。在这个视频协议，我们将演示如何通过阴性选择分离从人体血液中的NK细胞，利用干细胞技术的RosetteSep套件。该过程包括获取人外周血（下一个机构审查委员会批准的协议，以保护人类受试者），将缺席对NK细胞标记结合的抗体，鸡尾酒混合，但目前所有其他周边目前的单核细胞血液（例如，T淋巴细胞，单核细胞...).在鸡尾酒中的抗体结合向血型糖蛋白A对红细胞的抗体。因此，将所有不需要的细胞和红细胞被困联合体。然后稀释血和抗体鸡尾酒混合，是上Histopaque梯度重叠，离心。 NK细胞（> 80％的纯），可以收集Histopaque和稀释后的血浆之间的接口。类似的鸡尾酒为其他的细胞群，如人类T淋巴细胞，浓缩。
一个详细的协议，包括与每个玫瑰花九月隔离鸡尾酒，是对干细胞技术的网站 1可在网上。
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使用这种技术孤立的细胞可进一步分为不同的人群，使用其他细胞分离试剂盒。
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| Name | Type | Company | Catalog Number | Comments |
| Rosette Sep cocktail for human NK cells | Reagent | Stem Cell Technologies | 15065 | |
| PBS, steile, 1x | Reagent | GIBCO, by Life Technologies | 14040-058 | |
| Fetal calf serum (FCS, FBS) | Reagent | GIBCO, by Life Technologies | 16140-071 | |
| Histopaque-1077 | Reagent | Sigma-Aldrich | 10771 | |
| PBS supplemented with 2% FCS and Histopaque solutions must be brought to room temperature before the begining of the procedure. | ||||
1. Procedures and instruction manuals. http://www.stemcell.com/technical/technicalrsproc.aspx
