You are here:
AUTOFLUORESCENCE IN PRIMARY RAINBOW TROUT HEPATOCYTES INTERFERES WITH MEASUREMENT OF OXIDATIVE ACTIVITY VIA THE EXOGENOUS PROBE, DCF, BUT PROVIDES INTRINSIC MEASURE OF CELLULAR OXIDATIVE STATE
Citation:
Henry, T. R., R. D. Johnson, D B. Lothenbach, AND P. K. Schmieder. AUTOFLUORESCENCE IN PRIMARY RAINBOW TROUT HEPATOCYTES INTERFERES WITH MEASUREMENT OF OXIDATIVE ACTIVITY VIA THE EXOGENOUS PROBE, DCF, BUT PROVIDES INTRINSIC MEASURE OF CELLULAR OXIDATIVE STATE. Presented at SOT Annual Meeting, New Orleans, LA, March 14-18, 1999.
Description:
The compound 2', 7'-dichlorodihydrofluoroscein diacetate is a probe commonly used to detect oxidative activity in live cells. Studies were undertaken to measure reactive oxygen species generated in freshly isolated rainbow trout hepatocytes exposed to a variety of redox cycling componds, including 1,4-napthoquinone (NQ) and diquat (DQ). During our studies we found unusually high background levels of fluorescence in control, NQ- and DQ- treated cells loaded with DCF-DA. Similar levels of fluorescence were observed in freshly isolated cells which were not loaded with DCF-DA, indicating that the fluorescence is not due to the oxicized metabolite, DCF. It was determined that the background fluorescence apparent immediately upon isolation correlates with cell density, indicating that it is endogenous autofluorescence. The fluorescence was observed to increase with time in culture up to 24 hours post-isolation, reaching a maximum fluorescence up to an equivalent 50 pmol DCF. This temporal change in autofluorescence confounds interpretation of measurements of DCF fluorescence as an indicator of oxidative activity. However, the autofluorescence itself may be a useful endogenous indicator of the autofluorescence using multi-photon excitation microscopy indicates that it is consistent with previous reports of autofluorescence of flavoproteins and pyridine nucleotides in mammalian cells. Exploitation of these intrinsic oxidative activity in future mechanistic studies of cellular responses to oxidants in rainbow trout heptocytes.