Streamline Your Oxidative Stress Measurements

CellROX® Green and CellROX® Orange Reagents With Simple Workflows

(See a complete list of products discussed in this article.)

The generation of reactive oxygen species (ROS) is inevitable in healthy aerobic organisms. ROS production is mediated by auto-oxidation, photochemical reactions, and various enzymes (e.g., mitochondrial respiration, cytochrome P450, NADPH oxidase, and others), and is kept in check by the cell’s detoxification mechanisms. Oxidative stress occurs when there is an imbalance between ROS production and the cell’s ability to scavenge and detoxify them. This imbalance can cause transient or permanent oxidative damage in nucleic acids, proteins, and membrane lipids. It is also associated with the progression of several diseases, including inflammation, atherosclerosis, and age-related degenerative disorders.

Here we describe three cell-permeant, fluorogenic CellROX® reagents—CellROX® Deep Red reagent and the new CellROX® Green and CellROX® Orange reagents—for the detection and quantitation of ROS in live cells.

Detecting Oxidative Stress in Cells

The three CellROX® reagents form a set of photostable fluorogenic probes for measuring oxidative stress in live cells. These ROS sensors are compatible with multiple platforms, including microscopy (Figure 1), high-content imaging (Figure 2), flow cytometry, and microplate-based fluorometry, as well as with the FLoid® Cell Imaging Station and the  Attune® Acoustic Focusing Cytometer. Detecting ROS by conventional fluorescence microscopy or high-content imaging and screening offers several advantages over other experimental techniques. These imaging methods allow spatial and temporal resolution of ROS production, and also enable multiplex analysis with other measures of cell health and function.

The CellROX® Family of Oxidative Stress Sensors

In addition to CellROX® Deep Red reagent, we now offer two new CellROX® reagents for measuring oxidative stress in live cells (Table 1). CellROX® Green reagent (Figure 1, Figure 2) is a weakly fluorescent, cell-permeant dye that exhibits bright green fluorescence upon reaction with ROS and subsequent binding to DNA (excitation/emission maxima = 485/520 nm). This reagent is compatible with aldehyde-based fixatives as well as detergent permeabilization, making it well suited to subsequent antibody-based analysis.

CellROX® Orange reagent (Figure 1) is a nonfluorescent, cell-permeant dye that exhibits bright orange fluorescence upon reaction with ROS (excitation/emission maxima = 545/565 nm). While it is not compatible with aldehyde-based fixatives, CellROX® Orange reagent can be easily multiplexed with other fluorescent live-cell probes, including GFP, CellEvent® Caspase-3/7 Green detection reagent, Image-iT® DEAD Green viability stain, MitoTracker® Green FM and MitoTracker® Deep Red FM reagents, and NucBlue® live cell stain.

All three CellROX® reagents produce consistently bright and photostable fluorescence that can be detected by both traditional microscopy and high-content imaging to efficiently image and quantitate cellular oxidative stress. The signal that is generated with these reagents is specific for reactive oxygen species detection, as shown by the reduction in signal when cells are pre-incubated with N-acetyl cysteine (Figure 2).


Detection of ROS using fluorescence microscopy  
Figure 1. Detection of ROS using fluorescence microscopy. Human aortic smooth muscle (HASM) cells were plated in 35 mm glass-bottom dishes (MatTek) and left untreated as the control or treated with 500 nM angiotensin II for 4 hr at 37°C. Hoechst® 33342 and either CellROX® Green reagent (top row) or CellROX® Orange reagent (bottom row) were added for the last 30 min of incubation. The cells were then washed 3 times with PBS and imaged on a Zeiss® Axiovert inverted microscope using a 40x objective. An increase in CellROX® signal was observed after angiotensin II treatment, indicating an increase in oxidative stress in the treated cells.


Detection of ROS using fluorescence microscopyFigure 2. Detection of ROS using high-content imaging. Bovine pulmonary artery endothelial (BPAE) cells, plated in 96-well plates, were left untreated or treated with 100 µM menadione for 1 hr at 37°C to induce oxidative stress. In addition, a subset of control and menadione-treated wells also received 50 µM N-acetyl cysteine (NAC), an ROS scavenger. Cells were then stained with 5 µM CellROX® Green reagent and Hoechst® 33342 in complete medium for 30 min at 37°C, washed with PBS, and imaged on a Thermo Scientific Cellomics® ArrayScan® VTI. The decreased fluorescence seen in the presence of NAC confirms that the CellROX® Green signal is due to the presence of ROS in the samples.

Table 1. Features of several fluorogenic oxidative stress sensors.

CellROX® Green* CellROX® Orange* CellROX® Deep Red*

Ex/Em max (nm)† 485/520 545/565 644/665 504/529 518/605
Can be added to complete media Yes Yes Yes No Yes
Aldehyde fixable Yes No Yes No No
Detergent resistant Yes No No No No
Photostable ++ ++++ +++ + NA
Multiplexable Yes Yes Yes Yes No‡
GFP compatible No Yes Yes No No
RFP compatible Yes No Yes Yes No
Cat. No. C10444 C10443 C10422 D399 D11347
Quantity 5 x 50 μL 5 x 50 μL 5 x 50 μL 100 mg 10 x 1 mg
*The three CellROX® reagents are available individually or in a variety pack, which provides 50 μL of each reagent. †Excitation and emission maxima in nm for the oxidized reagent, in some cases bound to dsDNA. ‡The fluorescence emission spectra of oxidized DHE bound to DNA is very broad, making it difficult to multiplex with other fluorescent probes. H2DCFDA = 2′,7′-dichlorodihydrofluorescein diacetate. DHE = dihydroethidium. NA = data not available.

Simplify Your Workflows

Unlike the traditional dihydrodichlorofluorescein dyes (e.g., H2DCFDA), the CellROX® Green, CellROX® Orange, and CellROX® Deep Red reagents can be added directly to media containing serum, streamlining your experiments. Moreover, all three CellROX® reagents show superior photostability when compared with H2DCFDA (pdf from BioProbes 65). This photostability helps ensure more reliable and reproducible data and is particularly important for fluorescence imaging over time.

Take Advantage of Multiplexing

The CellROX® ROS sensors reliably measure oxidative stress and are compatible with a multitude of experimental platforms. With the introduction of additional fluorescent colors, CellROX® reagents are now even easier to combine with other live-cell probes for a more complete multicolor analysis of cell health.


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