Fluorescent Estimation of H_2O_2-Induced Changes in Cell Viability and Cellular Nonprotein Thiol Level of Dissociated Rat Thymocytes

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Author(s)

    • CHIKAHISA Lumi
    • Laboratory of Cell Signaling (Pharmacology), Faculty of Integrated Arts and Sciences, The University of Tokushima
    • OYAMA Yasuo
    • Laboratory of Cell Signaling (Pharmacology), Faculty of Integrated Arts and Sciences, The University of Tokushima
    • OKAZAKI Eisuke
    • Laboratory of Cell Signaling (Pharmacology), Faculty of Integrated Arts and Sciences, The University of Tokushima
    • NODA Katsuhiko
    • Laboratory of Cell Signaling (Pharmacology), Faculty of Integrated Arts and Sciences, The University of Tokushima

Abstract

We have developed a procedure to simultaneously estimate cell viability and the cellular level of nonprotein thiol (presumably glutathione) using two fluorescent dyes, 5-chloromethylfluorescein (5CMF) and ethidium, and rat thymocytes. Diethylmaleate and <I>N</I>-ethylmaleimide reduced, respectively, the intensity of 5CMF fluorescence to 0.23 and 0.1, relative to the control. Incubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, decreased the intensity of 5CMF fluorescence to 0.61. Results indicate that 5CMF fluorescence can be attenuated by agents that decrease the level of cellular nonprotein thiols, suggesting that 5CMF fluorescence is utilized for estimating the level of cellular glutathione. Hydrogen peroxide (10 μM to 3 mM) reduced the intensity of 5CMF fluorescence in a dose-dependent manner and increased the number of thymocytes stained with ethidium (presumably dead cells or cells with compromised membranes) at concentrations of 300 μM or greater. Reduction of cellular glutathione level seems to precede cell death in which oxidative stress is involved.

Journal

  • The Japanese Journal of Pharmacology

    The Japanese Journal of Pharmacology 71(4), 299-305, 1996-08-01

    The Japanese Pharmacological Society

References:  13

Cited by:  7

Codes

  • NII Article ID (NAID)
    10008189252
  • NII NACSIS-CAT ID (NCID)
    AA00691188
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    00215198
  • Data Source
    CJP  CJPref  J-STAGE 
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