Title

A novel sulfhydryl-sensing fluorescent probe to monitor the redox status of intracellular compartments

Aurora Affiliations

Sheikh Kalifa bin Hamad Al Thani Center for Integrative Research on Cardiovascular Aging

Presentation Notes

Presented at 2014 Aurora Scientific Day, Milwaukee, WI

Abstract

Background: Sensing of intracellular redox state is important for detecting the effect of disease and therapeutics agents. However, a reliable assay that can simultaneously provide information about the redox state of intracellular compartments is currently not available. Such an assay will improve the detection of abnormal metabolic state and evaluate the impact of therapeutics.

Purpose: To test the responsiveness of DSSQ1 (fluorescein- Donor tethered via a disulfide [S-S] to a para-methyl red Quencher), a novel sulfhydryl-sensing fluorescence probe, and monitor its intracellular distribution under oxidative and reduced conditions.

Methods: Fibroblasts grown in culture were treated with redox sensor DSSQ1, and its intracellular distribution and localization was assessed using confocal fluorescent microscopy. Localization of DSSQ1 within mitochondria, lysosomes and nuclei was confirmed using specific fluorescent dyes – TMRM for mitochondria, LysoTracker® Red for lysosomes and Hoechst 33342 for nuclei.

Results: Under the normal conditions, the green fluorescence of DSSQ1 was localized to the cytosol, lysosomes, nuclear membrane and within mitochondria. Oxidative stress (extracellular H2O2, 100 μM) significantly decreased the loading efficiency of the redox sensor DSSQ1 into the fibroblasts, while reducing agent (extracellular N-acetyl cysteine, 10 mM), which is known to increase intracellular levels of glutathione and cytoplasmic redox state, enhanced the uptake of DSSQ1.

Conclusion: The chemical structure of DSSQ1 allows permeability of compound without losing viability of cells. The compound is distributed within the cytoplasm, and localizes to lysosomes, mitochondria and nuclear membrane, but excluded from the nuclei. DSSQ1 accumulation is affected by redox status of cells and could be used to monitor the redox status of the cell.

Document Type

Abstract