FIgure 1. Reactive oxygen species (ROS) detected by flow cytometry. (A) ROS levels detected by the CellROX Deep Red Reagent are decreased in TBHP-treated Jurkat cells with pretreatment of cultures using NAC. The cells treated with the oxidant TBHP (red) have increased staining with the CellROX Deep Red Reagent, compared to the cells pretreated with NAC (blue) and the control cells (green). (B, C) CellROX Deep Red Reagent can be used in conjunction with SYTOX Blue Dead Cell Stain to differentiate live stressed cells from dead cells. Jurkat cells were treated with (B)PBS or (C) 200 μM TBHP for 30 minutes before labeling with the CellROX Deep Red Flow Cytometry Assay Kit. Note that the treated cells (C) have a higher percentage of cells under oxidative stress than the basal level of ROS observed in control cells (B).
Three Invitrogen Molecular Probes fluorogenic reagents—CellROX Deep Red, CellROX Green and CellROX Orange —have been developed for the detection and quantitation of reactive oxygen species (ROS) in live cells. Each reagent is cell-permeant and is nonfluorescent or very weakly fluorescent in the reduced state. Upon oxidation, the reagents exhibit strong fluorescence and remain localized within the cell.
These ROS sensors are compatible with multiple platforms, including flow cytometry, microscopy, high-content imaging and microplate-based fluorometry, as well as with the Invitrogen Attune Acoustic Focusing Cytometer and the Invitrogen FLoid Cell Imaging Station.
CellROX Reagents offer significant advantages over the traditional dihydrodichlorofluorescein dyes (e.g.,H2DCFDA) such as:
- Fluorogenic probes—CellROX reagents become brightly fluorescent when oxidized in cells, allowing sensitive detection
- Multicolor compatibility—minimal overlap with fluorophores excited by other laser lines, allowing easy multiplexing with other reagents
- Simple—cells can be stained in complete media or other appropriate buffers; no need for serum-free media
- Fixable—CellROX Green and CellROX Deep Red Reagents retain signal following formaldehyde fixation (CellROX Orange Reagent is not compatible with fixation)
- Flexibility—Reagents are available in 3 different fluorescent colors, making experimental design more convenient
A comparison of these features can be seen in Table 1. To see more products for ROS detection, see our Oxidative Stress selection guide.
Table 1. Features of several fluorogenic oxidative stress sensors.
Features | H2DCFDA | DHE | |||
|---|---|---|---|---|---|
| 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 |
| * 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. | |||||
In addition to the stand alone reagents, we have developed kits validated for use in flow cytometry that include:
- An antioxidant (N-acetylcysteine [NAC], to serve as a negative control)
- An oxidant (tert-butyl hydroperoxide [TBHP], to serve as a positive control)
- A SYTOX dead cell stain compatible with the CellROX reagent
When the CellROX reagent is used together with the Invitrogen SYTOX Red Dead Cell Stain (or SYTOX Blue Dead Cell Stain with CellROX Deep Red Reagent), oxidatively stressed and nonstressed cells are reliably distinguished from dead cells by flow cytometry.
All three CellROX reagents produce consistently bright and photostable fluorescence that can be detected by both traditional microscopy (Figure 2) and high-content imaging (Figure 3) 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).
Figure 2. 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.
Figure 3. 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.
