Shedding Light on Oxidative Stress
CellROX™ Deep Red Reagent For ROS Detection
Generation of reactive oxygen species (ROS) is inevitable for aerobic organisms and, in healthy cells, occurs at a controlled rate. Under conditions of oxidative stress, ROS production is dramatically increased, resulting in subsequent alteration of membrane lipids, proteins, and nucleic acids. Oxidative damage of these biomolecules is associated with aging as well as with a variety of pathological events, including atherosclerosis, carcinogenesis, ischemic reperfusion injury, and neurodegenerative disorders. Oxidative stress can be caused by many different intrinsic and extrinsic pathways that are mediated either by mitochondrial respiration or by membrane-bound NADPH oxidases [1–3]. CellROX™ Deep Red reagent is a new fluorogenic probe to measure oxidative stress in cells using conventional fluorescence microscopy (Figure 1), high-content imaging, microplate fluorometry, or flow cytometry.
Figure 1. Imaging oxidative stress with CellROX™ Deep Red reagent. Human osteosarcoma (U2OS) cells were treated with 100 µM menadione for 1 hr to induce oxidative stress, and then stained with 5 µM CellROX™ Deep Red reagent, 5 µg/mL of CellMask™ Orange plasma membrane stain, and 1 µM SYTO Green fluorescent nuclear stain for 30 min at 37°C. The cells were washed 3 times with HBSS before imaging.
Simple and Flexible Workflow
The cell-permeant CellROX™ Deep Red dye is nonfluorescent in a reduced state and produces bright near-infrared fluorescence upon oxidation (Figure 2). The resulting fluorescence can be measured using fluorescence imaging, high-content imaging, microplate fluorometry, or flow cytometry. In addition to allowing ROS detection in live cells, the signal is retained after formaldehyde fixation. The flexibility of the CellROX™ Deep Red reagent results in improved workflows compared to ROS detection based on classic dyes (Table 1). The staining workflow is simple (Figure 3), and the reagent can be applied to cells in complete growth media.
Figure 2. Fluorescence properties of the CellROX™ Deep Red reagent. (A) Fluorescence excitation and emission spectra. The dye has peak excitation and emission at 640 nm and 665 nm, respectively. (B) Mechanism of action. The reagent is cell permeant and nonfluorescent in a reduced state, and produces intense fluorescence upon oxidation.
Table 1. Comparison of ROS Detection Methods.
|Dihydrodichlorofluorescein, diacetate (H2DCFDA)||Dihydroethidium (DHE)||CellROX™ Deep Red reagent|
|Labeling in complete media
Figure 3. Simple workflow for CellROX™ Deep Red reagent staining of adherent cells. For flow cytometry, cells must be detached before analysis. CellROX™ Deep Red reagent will also work with suspension cells.
Detection with Traditional Fluorescence Microscopy or High-Content Imaging
Figure 4. Detection of ROS with traditional fluorescence microscopy. Human aortic smooth muscle cells were plated in 35 mm glass bottom dishes (MatTek) and treated with 500 nM angiotensin II for 4 hr at 37°C. CellROX™ Deep Red reagent (5 µM) and Hoechst 33342 (blue) were added for the last 30 min of compound incubation. The cells were washed 3 times with PBS and imaged on a Zeiss Axiovert inverted microscope using a 40x objective. An increase in CellROX™ Deep Red signal (red) was observed after angiotensin II treatment, indicating an increase in oxidative stress in these cells. (A) Control cells. (B) Cells treated with angiotensin II.
Figure 5. Detection of ROS using high-content imaging. Bovine pulmonary artery endothelial (BPAE) cells or RAW macrophage cells were plated in 96-well plates. (A) BPAE cells were treated with or without 100 µM menadione for 1 hr at 37°C (left and middle panels). The superoxide scavenger MnTBAP (100 µM) was added to several of the control and menadione-treated wells for the last 30 min of incubation (right panel). (B) RAW cells were treated with or without 500 ng/mL of LPS and 150 nM diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, for 24 hr at 37°C. The cells described in (A) and (B) were then stained with 5 µM CellROX™ Deep Red reagent in complete medium for 30 min at 37°C, then washed with PBS and analyzed on a Thermo Scientific Cellomics ArrayScan VTI. MnTBAP or DPI treatment inhibited oxidative stress caused by menadione or LPS, respectively, confirming that the signal was due to ROS induced by these compounds. ***a values were significantly different from controls, with P ≤ 0.0001; ***b values were significantly different from drug-treated cells, with P ≤ 0.0001.
Easy Multiplexing with Other Cell Health Markers
CellROX™ Deep Red reagent is multiplexable with other fluorescent probes, including those for cell health such as the Image-iT DEAD Green™ viability stain (Figure 6A) and the CellEvent™ Caspase 3/7 Green detection reagent for apoptosis (Figure 6B), providing convenience in measuring multiple parameters of cell health.
Figure 6. Multiplex measurements of oxidative stress with cytotoxicity or apoptosis. HepG2 cells were plated in 96-well plates and treated with 50 µM nefazodone for 24 hr at 37°C. During the last 30 minutes of treatment, the cells were stained with 5 µM CellROX™ Deep Red reagent (red), Hoechst 33342 (blue), and 20 nM Image-iT DEAD Green™ viability stain (green) (A) or 5 µM CellEvent™ Caspase 3/7 Green detection reagent (green) (B). The cells were washed 3 times with PBS and analyzed on a Thermo Scientific Cellomics ArrayScan VTI. In some nefazodone-treated cells, an increase in Image-iT DEAD Green™ signal indicated plasma membrane permeability, a marker for cytotoxicity (A), while an increase in CellEvent™ caspase 3/7 signal indicated apoptotic cells (B). In both samples, nefazodone caused oxidative stress. Fold changes are ratios of mean signal intensities of treated samples to mean signal intensities of control samples. *** Mean signal intensities were significantly different from controls, with P ≤ 0.0001; ** Mean signal intensities were significantly different from controls, with P ≤ 0.001.
Simple, Reliable ROS Detection
The CellROX™ Deep Red reagent reliably measures oxidative stress by traditional fluorescence microscopy, automated high-content imaging, flow cytometry, and microplate fluorometry. The probe can be used in conjunction with other live-cell dyes and GFP but is also formaldehyde-fixable, providing excellent utility in multiplex fluorescence assays.