Oxidative Stress in Bacteria Measured by Flow Cytometry

Authored by Daniel Manoil

The CellROX® Deep Red flow cytometry kit (Life technologies) has been developed for the detection of oxidative stress in mammalian cells. It combines a ROS sensitive fluorophore (CellROX® Deep Red) and a "viability” dye (SYTOX® Blue) to allow the detection of non-oxidized, oxidized and damaged cells. The present study investigated the application of these markers to Enterococcus faecalis and Fusobacterium nucleatum subjected to oxidative stress. An optimal concentration of CellROX® has been determined on Enterococcus faecalis and Fusobacterium nucleatum exposed to oxidative stress (H2O2). Bacteria have been exposed to various H2O2 concentrations and labeled with CellROX® to verify that fluorescence increased along with oxidative stress. Also, bacteria exposed to H2O2 were double stained with CellROX® and SYTOX® Blue and analyzed by flow cytometry. The optimal concentration of CellROX® Deep Red was 4^M for both strains. Fluorescence of bacteria labeled with 4^M of CellROX® Deep Red increased accordingly with the oxidative stress applied. Flow cytometry analysis of double stained samples showed bacteria subpopulations with increased CellROX® signal when stressed, and higher SYTOX® Blue uptake under higher oxidative stress. Results indicate that CellROX® Deep Red can be applied to measure oxidative stress in E. faecalis and F. nucleatum. The combination of CellROX® and SYTOX® Blue allowed the discrimination of non-oxidized, oxidized and damaged bacteria.

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