전자저널 > 전자저널 > Quantification of nitric oxide concentration in the hemocytes of Manila clam

Quantification of nitric oxide concentration in the hemocytes of Manila clam

Ki-Woong Nam, Hyun-Sung Yang and Kyung-Il Park

Department of Aquatic Life Medicine, Kunsan National University, Gunsan, Jeonbuk 573-701, Republic of Korea East Sea Research Institute, Korea Institute of Ocean Science and Technology (KIOST), Uljin, Kyungbuk 767-813, Republic of Korea

Nitric oxide (NO) is an important intra-intercellular signaling molecule that regulates many physiological processes and participates in the development some pathological conditions in animals. In this study, we compared different methods for determining NO concentration in the hemocytes of Manila clam Ruditapes philippinarum. For measuring the intracellular NO levels, we used the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA), and the quantification methods that were compared were based on image analysis, spectrophotometry, and flow cytometry. NO concentration could be determined using all the 3 methods, and the concentration varied significantly depending upon the presence of NO regulators in the hemocytes; NO concentration increased in the presence of L-arginine, while it decreased in the presence of N-nitro-L-arginine methyl ester. In particular, it is found that estimation of NO using a flowcytometry is more economical, reliable and accurate compared to image analysis and spectrophotometry. Accordingly we believe that determining NO concentration by using flowcytometry will be useful in evaluating physiological and pathological conditions in marine bivalves.

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