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Author:
Date:
2016-10-05
[Abstract] The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species (ROS) are strictly regulated by the cells. However, during stressful conditions such as oxidative stress, ROS levels increase causing damages to different molecules like DNA, lipids and proteins. Increased levels of ROS have been associated with a growing list of different diseases. In this protocol, we used MitoSOX and CellROX Green oxidative stress probes to label the intracellular ROS and detect the fluorescence using cell sorting and confocal analyses.
[摘要] 自由基的产生是正常细胞代谢的结果。 自由基涉及无数不同的细胞和生物学功能，例如信号传导，增殖，细胞死亡，衰老，炎症等。 在生理条件下，活性氧（ROS）的水平被细胞严格调节。 然而，在应激条件例如氧化应激期间，ROS水平增加导致对不同分子如DNA，脂质和蛋白质的损伤。 增加的ROS水平与不断增加的不同疾病列表相关联。 在这个协议中，我们使用MitoSOX和CellROX绿色氧化应激探针标记细胞内ROS和使用细胞分选和共聚焦分析检测荧光。
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Author:
Date:
2013-09-20
[Abstract] Calcium influx is one of the key signaling events upon stimulation of T cell receptors (TCR) and plays an important role for T cell activation, proliferation, and differentiation. Phorbol myristate acetate (PMA) and calcium ionophore ionomycin are commonly utilized as stimulants in a variety of immunologic assays including T cell activation. PMA is a protein kinase C (PKC) activator, resulting in the activation of Ras, a small GPTase. When PMA and ionomycin are used together, TCR signaling downstream of PKC and Ras can be activated without activation of TCR-triggerd signaling events. This protocol describes the flow cytometry analysis of intracellular calcium influx in T cells stimulated with PMA and ionomycin.
[摘要] 钙流入是刺激T细胞受体（TCR）的关键信号事件之一，并且对T细胞活化，增殖和分化起重要作用。 佛波醇肉豆蔻酸乙酸酯（PMA）和钙离子载体离子霉素通常在多种免疫测定（包括T细胞活化）中用作刺激剂。 PMA是蛋白激酶C（PKC）激活剂，导致Ras（一种小GPTase）的活化。 当PMA和离子霉素一起使用时，PKC和Ras下游的TCR信号传导可以在没有激活TCR-触发信号事件的情况下被激活。 该协议描述了用PMA和离子霉素刺激的T细胞中的细胞内钙流入的流式细胞术分析。
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