Fluorescence Imaging of Oxidative Stress in Live Cells

Oxidative stress is caused by elevated production or reduced detoxification of reactive oxygen species (ROS) in cells. ROS can directly damage the cell’s DNA, proteins, and lipids, and increased oxidative stress is associated with both apoptotic and autophagic cell death. Importantly, increased oxidative stress and dysregulation of cell death mechanisms have been linked with aging as well as several disease states.

Robust ROS Assays With Simple Workflows

The cell-permeant CellROX™ Deep Red reagent is a fluorogenic ROS sensor. In its reduced state, CellROX™ Deep Red reagent is nonfluorescent; however, upon oxidation by ROS, this reagent exhibits bright near-infrared fluorescence that serves as a direct measurement of the ROS levels in live cells. With excitation/emission maxima of 640/665 nm, the fluorescence of the oxidized CellROX™ Deep Red reagent can be detected using fluorescence microscopy, high-content imaging, microplate fluorometry, or flow cytometry, using the same filters and settings you use for Alexa Fluor® 647 and Cy®5 dyes.

CellROX™ Deep Red reagent represents a significant breakthrough in the measurement of ROS in live cells. Not only does it provide sensitive detection of ROS, but CellROX™ Deep Red reagent is also exceptionally simple to use. This ROS sensor can be applied directly to live cells in complete growth media. After a 30-minute incubation and three buffer washes, the stained cells are ready to be analyzed. CellROX™ Deep Red staining survives formaldehyde fixation—unlike other common ROS sensors such as dihydrodichlorofluorescein diacetate (H2DCFDA)—but is not compatible with detergent-based permeabilization protocols.

Superior Photostability

A particular advantage of CellROX™ Deep Red reagent over H2DCFDA is its superior photostability (Figure 1). The extraordinary photostability of CellROX™ Deep Red reagent ensures more reliable and reproducible data and is particularly important for fluorescence imaging over time.

CellROX™ Deep Red reagent

Figure 1. Photostability analysis of CellROX™ Deep Red reagent.
Human osteosarcoma (U2OS) cells were treated with 100 μM menadione for 1 hr to induce oxidative stress and then incubated for 30 min at 37°C either with 5 μM CellROX™ Deep Red reagent in complete growth medium or with 5 μM H2DCFDA in HBSS. Cells were washed 3 times with HBSS before imaging. Samples were continuously illuminated on a Zeiss® Axiovert® inverted microscope for 14 sec, and photographs were taken with exposure times of 500 msec and 150 msec for CellROX™ Deep Red reagent and H2DCFDA, respectively. The fluorescent signal intensities were quantitated using SlideBook™ 5 software, represented as normalized intensity values, and plotted against time of illumination.

Get a More Complete Picture of Cellular Oxidative Stress

CellROX™ Deep Red reagent can be used with other live-cell probes and with GFP, providing many options for multiplex fluorescence assays. We offer several other ROS sensors, including the fluorescein derivatives APF and HPF, which detect hydroxyl radicals, peroxynitrite anion, and singlet oxygen (APF also detects hypochlorite anion). For the detection of mitochondrial superoxide, we offer the MitoSOX™ Red indicator.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.