Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method

详细信息	查看全文 | 推荐本文 |

• 作者：Reena V. Kartha^a ; ^b ; ^{rvkartha@umn.edu} ; Jie Zhou^a ; ^b ; Laurie B. Hovde^a ; ^b ; Belinda W.Y. Cheung^c ; Henning Schrö ; der^c
• 关键词：Gaseous mediators ; Hydrogen sulfide ; Measurement ; Trapping ; Donors ; Inhibitors
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：1 April, 2012
• 卷：423
• 期：1
• 页码：102-108
• 文件大小：599 K

摘要

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 渭M. Following a 24-h incubation of endothelial-like or vascular smooth muscle cells with 50 渭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.