Visualization of the compartmentalization of glutathione and protein‐glutathione mixed disulfides in cultured cells

Using FACS analysis of anti-GSH antibody-stained Jurkat T lymhocytes, we demonstrated population variations in the cellular compliment of GSH and protein-GSH mixed disulfides, formed in response to diamide

Therese Söderdahl; Mari Enoksson; Mathias Lundberg; Arne Holmgren; Ole P Ottersen; Sten Orrenius; George Bolcsfoldi; Ian A Cotgreave

2003

Scholarcy highlights

  • Fluorescence microscopy of A549 cells stained with a glutathione-specific polyclonal antibody displayed uniform staining of the perinuclear cytosol, with the nuclear region apparently lacking GSH staining
  • Immunocytochemical staining of A549 cells with an anti-GSH antibody and subsequent visualization by confocal microscopy revealed that the concentration of GSH in the cells is unevenly distributed across the focal plane
  • Cytochemical staining of the cells with mercury orange showed a low nuclear:cytosolic ratio of GSH staining in A549 cells
  • The specificity of staining for free GSH in each staining procedure is illustrated in Figure 2, where it is clear that preincubation of A549 cells with buthionine sulfoximine drastically diminished the intensity of microscopic images from the subsequently stained cells
  • Methods have been developed to assess intracellular GSH utilizing glutathione S-transferase-mediated conjugation of substrates such as monochlorobimane to yield fluorescent GSH conjugates that are amenable to analysis, either by microscopy or by FACS
  • Cells were plated in a 12-well plate and grown to 95% confluency, and groups were exposed with one of the following treatments: untreated, cells fixed with methanol:acetone for 5 min; cells fixed as above and washed with phosphate-buffered saline once; and cells fixed and washed as above and fixed again with methanol:acetone for 5 min
  • The first is related to the dynamic nature of the labeling, being enzyme-dependent, and the second is due to the production of potential artifacts due to rapid intracellular distribution of conjugates
  • The data generated from the analysis of mitochondrial GSH in intact cells clearly suggested that there is a wide variation in GSH contents within a population of mitochondria displaying similar granularity

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