Production of Superoxide in Bacteria Is Stress- and Cell State-Dependent: A Gating-Optimized Flow Cytometry Method that Minimizes ROS Measurement Artifacts with Fluorescent Dyes

Abstract

The role of reactive oxygen species (ROS) in microbial metabolism and stress response has emerged as a major theme in microbiology and infectious disease. Reactive fluorescent dyes have the potential to advance the study of ROS in the complex intracellular environment, especially for high-content and high-throughput analyses. However, current dye-based approaches to measuring intracellular ROS have the potential for significant artifacts. Here, we describe a robust platform for flow cytometric quantification of ROS in bacteria using fluorescent dyes, with ROS measurements in 10s-of-1000s of individual cells under a variety of conditions. False positives and variability among sample types (e.g., bacterial species, stress conditions) are reduced with a flexible four-step gating scheme that accounts for side- and forward-scattered light (morphological changes), background fluorescence, DNA content, and dye uptake to identify cells producing ROS. Using CellROX Green dye with Escherichia coli, Mycobacterium smegmatis, and Mycobacterium bovis BCG as diverse model bacteria, we show that (1) the generation of a quantifiable CellROX Green signal for superoxide, but not hydrogen peroxide-induced hydroxyl radicals, validates this dye as a superoxide detector; (2) the level of dye-detectable superoxide does not correlate with cytotoxicity or antibiotic sensitivity; (3) the non-replicating, antibiotic tolerant state of nutrient-deprived mycobacteria is associated with high levels of superoxide; and (4) antibiotic-induced production of superoxide is idiosyncratic with regard to both the species and the physiological state of the bacteria. We also show that the gating method is applicable to other fluorescent indicator dyes, such as the 5-carboxyfluorescein diacetate acetoxymethyl ester and 5-cyano-2,3-ditolyl tetrazolium chloride for cellular esterase and reductive respiratory activities, respectively. These results demonstrate that properly controlled flow cytometry coupled with fluorescent probes provides precise and accurate quantitative analysis of ROS generation and metabolic changes in stressed bacteria.

Foraminiferal Ultrastructure: A perspective From Fluorescent and Fluorogenic Probes

“…This fluorogenic probe is almost nonfluorescent when in a reduced state and shows a bright fluorescent signal when it is oxidized. Thus, unstressed cells with a low production of ROS show a low fluorescence signal, while when the production of ROS increases due to an oxidative stress condition, cells show a higher fluorescence (McBee et al, ). This probe can be used in both living and fixed cells.…”

All three quinone species play distinct roles in ensuring optimal growth under aerobic and fermentative conditions in E. coli K12

“…To determine if mutant strains suffer from oxidative stress, flow cytometry with CellROX Green Reagent (Thermo Fisher Scientific) was applied, according to the protocol provided by the manufacturer and similar to the protocol of McBee [ 37 ]. After incubation of the second pre-culture without shaking overnight, the cells were centrifuged at 5000 x g for 20 min.…”

A Current Overview of the Biological and Cellular Effects of Nanosilver

“…Additionally, the cells, media, and the order of treatment used in doing the H 2 DCFDA assay can affect the outcome [ 77 ]. A more reliable method is to use a fluorescent dye and flow cytometry with proper gating to remove artifacts due to background fluorescence and cell debris [ 78 ]. CellROX fluorescent probes are useful for detecting •O 2 − and •OH.…”