Hydrogen sulfide (H2S) has received great attention as a third gaseous signal transmitter, following nitric oxide and carbon monoxide. In particular, H2S plays an important role in the regulation of cancer cell biology. Therefore, the detection of endogenous H2S concentrations within biological systems can be helpful to understand the role of gasotransmitters in pathophysiology. Although a simple and inexpensive method for the detection of H2S has been developed, its direct and precise measurement in living cells remains a challenge. In this study, we introduced a simple, facile, and inexpensive colorimetric system for selective H2S detection in, living cells,using a silver -embedded Nafion/polyvinylpyrrolidone (PVP) membrane. This membrane could be easily applied onto a polystyrene microplate cover. First, we optimized the composition of the coating membrane, such as the PVP/Nafion mixing ratio and AgNO3 concentration, as well as the pH of the Na2S (H2S donor) solution and the reaction time. Next, the in vitro performance of a colorimetric detection assay utilizing the silver/Nafion/PVP membrane was evaluated utilizing a known concentration of Na2S standard solution both at room temperature and at 37 degrees C in a 5% CO2 incubator. As a result, the sensitivity of the colorimetric assay for H2S at 37 C in the incubator (0.0056 Abs./mu M Na2S, R-2 = 0.9948) was similar to that at room temperature (0.0055 Abs./mu NM Na2S, R-2 = 0.9967). Moreover, these assays were less sensitive to interference from compounds such as glutathione, t-cysteine (Cys), and dithiothreitol than to the H2S from Na2S. This assay based on the silver/Nafion/PVP membrane also showed excellent reproducibility (2.8% RSD). Finally, we successfully measured the endogenous H2S concentrations in live C6 glioma cells by s-(5'-adenosyl)-L-methionine stimulation with and without Cys and L-homocysteine, utilizing the silver/Nafion/PVP membrane. In summary, colorimetric assays using silver/Nafion/PVP-coated membranes can be simple, robust, and reliable tools for the detection of H2S that can avoid the complicated and labor-intensive analytical approach used in conventional biology. In addition, we expect that this assay will demonstrate a powerful ability to study pathophysiological pathways that involve H2S. (C) 2017 Elsevier B.V. All rights reserved.