Measuring Intracellular Calcium Signaling in Murine NK Cells by Flow Cytometry
Measuring Intracellular Calcium Signaling in Murine NK Cells by Flow Cytometry
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DOI：
10.1007/978-1-60761-362-6_10
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发表时间：
2010-01-01
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影响因子：
--
通讯作者：
Campbell, Kerry S.
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文献类型：
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作者：
MacFarlane, Alexander W.;Oesterling, James F.;Campbell, Kerry S.
本章描述了一种方法，通过该方法可以使用配有紫外激光器的流式细胞仪在单个小鼠NK细胞中测量激活受体介导的钙信号。该方法的一个主要优点是，少数NK细胞群体和甚至更小的NK细胞亚群的钙反应可以从新鲜制备的总脾细胞的混合物中同时测量，而无需借助于先前的细胞分选或培养物中的扩增。简言之，收获细胞并染色以标记感兴趣的群体，然后用indo-1AM染料加载并在流式细胞仪上分析。在建立适当的基线后，用针对活化受体的生物素化抗体处理细胞，随后通过加入链霉亲和素交联。细胞内钙的增加通过测量当染料与钙结合时发生的indo-1发射光谱的位移来定量。
This chapter describes a method by which activating receptor-mediated calcium signaling can be measured in individual murine NK Cells Using a flow cytometer fitted with a UV laser. One major advantage of this method is that the calcium response of the minority NK cell population and even smaller NK cell subpopulations can be measured simultaneously from a mixture of freshly prepared total splenocytes without resorting to prior cell sorting or expansion in culture. Briefly, cells arc harvested and stained to mark the populations of interest, then loaded with indo-1 AM dye and analyzed on the flow cytometer. After an appropriate baseline is established, the cells are treated with a biotinylated antibody to activating receptors, which arc subsequently cross-linked by addition of streptavidin. The increase in intracellular calcium is quantified by measuring a shift in the indo-1 emission spectrum that takes place when the dye becomes bound to calcium.