Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method
Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method
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DOI：
10.1016/j.ab.2012.01.001
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发表时间：
2012-04-01
影响因子：
2.9
通讯作者：
Schroeder, Henning
中科院分区：
文献类型：
--
作者：
Kartha, Reena V.;Zhou, Jie;Schroeder, Henning
由于缺乏可靠的方法来准确测量体外产生的硫化氢(H_2S)，阻碍了对这种气体介质的生理学研究。目前的体外方法包括在与硫化氢释放化合物孵育后测量细胞培养液中的硫化氢。然而，这种方法是不准确的，因为硫化氢气体的寿命很短，因此无法检测到。为了克服这一点，我们采用了一种方法，采用了一种改进的琼脂层来立即捕获硫化氢，从而可以测量随着时间积累的硫化氢。在琼脂中捕获的硫化氢的数量是用原位亚甲基蓝测定的。我们能够检测到硫化氢钠(NaHS)在低至10mU的浓度下产生的硫化氢，在内皮样细胞或血管平滑肌细胞与50mU硫化氢(NaHS)孵育24小时后，我们能够回收比传统方法多一倍的硫化氢。在细胞培养中加入硫化氢释放化合物L-半胱氨酸和N-乙酰半胱氨酸后，细胞中的硫化氢含量呈浓度、时间和细胞系依赖性增加。总之，我们开发了一种改进的方法来量化体外产生的硫化氢。这种方法可以用来筛选化合物，以确定潜在的硫化氢供体和抑制剂用于治疗。(C)2012 Elsevier Inc.保留所有权利。
Lack of reliable methods to accurately measure hydrogen sulfide (H2S) produced in vitro has impeded research on the physiology of this gaseous mediator. Current in vitro methods involve measurement of H2S in cell culture media following incubation with H2S-releasing compounds. However, this method is inaccurate because H2S gas has a short life and thus evades detection. To overcome this, we have adapted a method that employs a modified agar layer to instantly trap H2S, allowing measurement of H2S accumulated with time. The amount of H2S trapped in the agar is quantified using an in situ methylene blue assay. We were able to detect H2S produced from sodium hydrogen sulfide (NaHS) added at concentrations as low as 10 mu M. Following a 24-h incubation of endothelial-like or vascular smooth muscle cells with 50 mu M NaHS, we were able to recover twice more H2S than conventional methods. When H2S-releasing compounds l-cysteine and N-acetylcysteine were added to the cell culture, the amount of H2S increased in a concentration-, time-, and cell line-dependent manner. In conclusion, we have developed an improved method to quantify H2S generated in vitro. This method could be used to screen compounds to identify potential H2S donors and inhibitors for therapeutic use. (C) 2012 Elsevier Inc. All rights reserved.