Matheu Melanie P, Cahalan Michael D
Department of Physiology and Biophysics, University of California, Irvine, CA, USA.
J Vis Exp. 2007(9):409. doi: 10.3791/409. Epub 2007 Nov 1.
Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are > or = 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!
从原始来源分离细胞是许多更复杂实验方案中的必要步骤。美天旎提供从多种生物体（包括人类、非人灵长类动物、大鼠以及如我们在此所述的小鼠）中分离细胞的试剂盒。基于磁珠的细胞分离可实现阳性选择（或细胞去除）以及阴性选择。在此，我们展示了对未接触过的或天然的CD4 +辅助性T细胞的阴性选择。使用此标准方案，我们通常可纯化出纯度≥96%的CD4 + / CD3 +细胞。本方案与发表于《可视实验期刊》第7期的“小鼠外周淋巴结的解剖和双光子成像”方案结合使用，用于纯化T细胞和其他细胞类型以用于成像目的的过继转移。尽管我们在本方案视频中未展示流式细胞术分析，但强烈建议通过流式细胞术使用适当抗体检查分离细胞的总体纯度。此外，我们展示了T细胞分离的非无菌方法。如果您特定的最终用户应用需要无菌细胞，请务必在标准无菌条件下于组织培养罩中进行所有展示的操作。感谢观看，祝您自己的实验顺利！
