Oxidative stress from a cell’s point of view
From CellROX® ROS sensors to Image-iT® and Click-iT® lipid peroxidation detection kits
The generation of reactive oxygen species (ROS) is a natural consequence of aerobic energy metabolism. Oxidative stress occurs when the cell’s antioxidant capacity is unable to keep pace with the production of ROS. Oxidative stress can lead to damage to the DNA, proteins, and lipids in the cell, and this damage has been linked to aging as well as to the progression of diseases such as atherosclerosis, neurodegenerative disorders, and cancers.
This article highlights three easy-to-use products for measuring various forms of oxidative stress by fluorescence microscopy, quantitative fluorescence microscopy (i.e., high-content imaging or HCI), automated microplate fluorometry, and flow cytometry. CellROX® reagents are fluorogenic indicators for the direct detection and quantitation of ROS in cells (Table 1). The Image-iT® Lipid Peroxidation Kit provides the reagents and protocol for real-time lipid peroxidation sensing in live cells; likewise, the Click-iT® Lipid Peroxidation Imaging Kit allows you to detect lipid peroxidation–derived protein modifications in fixed cells.
Table 1. Comparison of CellROX® reagents with conventional fluorogenic oxidative stress sensors.
|Features||CellROX® Green*||CellROX® Orange*||CellROX® Deep Red*||H2DCFDA||DHE|
|Ex/Em max (nm)†||508/525||545/565||644/665||504/529||518/605|
|Can be added to complete media||Yes||Yes||Yes||No||Yes|
|Compatibility with GFP or RFP||RFP||GFP||Both GFP and RFP||RFP||Neither GFP nor RFP|
|Stand-alone reagent—for imaging, microplate, and flow cytometry applications||C10444
5 x 50 μL
5 x 50 μL
5 x 50 μL
|Flow cytometry kit||C10492||C10493||C10491||—||—|
|*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 spectrum 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.|
Directly detect ROS in live cells with CellROX® reagents
Cell-permeant CellROX® reagents are nonfluorescent (or very weakly fluorescent) while in a reduced state and exhibit strong fluorescence when oxidized, making them straightforward and reliable indicators of ROS in live cells (Figure 1). CellROX® Green Reagent is a modified nucleic acid dye that, upon oxidation, binds to DNA, producing a green-fluorescent signal that is localized primarily in the nucleus and mitochondria. In contrast, the fluorescent signals from oxidized CellROX® Deep Red and CellROX® Orange Reagents are localized in the cytoplasm. The staining patterns of CellROX® Green and CellROX® Deep Red Reagents survive aldehyde fixation, and all three reagents are more photostable than traditional ROS indicators (e.g., H2DCFDA) (Table 1).The availability of three different fluorescent colors allows you to multiplex CellROX® reagents with a multitude of other cell stains and labeling reagents.
The stand-alone CellROX® reagents are supplied as ready-to-use DMSO solutions. These ROS sensors can be directly added to cells in complete growth medium or buffer. After a 30-minute incubation and brief wash, the cells are ready for analysis. CellROX® staining is compatible with multiple analysis platforms—including fluorescence microscopy [1,2], quantitative fluorescence microscopy (HCI), microplate-based fluorometry, and flow cytometry [3,4]—as well as with the EVOS® family of cell imaging stations, the Tali® Image-Based Cytometer, and the Attune® Acoustic Focusing Cytometer.
|Figure 1. ROS detection with CellROX® Deep Red Reagent. BPAE cells were (A) left untreated or (B) treated with 100 μM menadione, which causes oxidative stress, for 1 hr at 37°C. (C) The superoxide scavenger MnTBAP (100 μM) was added to some menadione-treated wells for the last 30 min of incubation. All cells were stained with CellROX® Deep Red Reagent for 30 min in complete growth medium at 37°C, washed with PBS, and analyzed on a Thermo Scientific® Cellomics® ArrayScan® VTI. Signal reduction in antioxidant-treated cells confirms ROS detection by CellROX® Deep Red Reagent.|
New CellROX® kits validated for flow cytometry
In addition to the stand-alone reagents, we have developed three CellROX® kits validated for use in flow cytometry. These kits provide a complete set of reagents for distinguishing oxidatively stressed cells, nonstressed cells, and dead cells (Figure 2), including:
- CellROX® Green, CellROX® Orange, or CellROX® Deep Red Reagent
- 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, color-matched with the CellROX® reagent
The CellROX® Flow Cytometry Assay Kits contain reagents that have been formulated to work well together and exhibit minimal overlap with fluorophores excited by other laser lines. You can use these flow cytometry kits to multiplex oxidative stress detection with other measures of cell structure or function, such as probes for mitochondrial membrane potential, cytoskeleton integrity, or apoptosis.
|Figure 2. ROS detection by flow cytometry. (A) ROS levels detected by the CellROX® Deep Red Reagent (provided in the CellROX® Deep Red Flow Cytometry Assay Kit) are decreased in oxidant-treated Jurkat cells with pretreatment of cultures using N-acetylcysteine (NAC). The cells treated with the oxidant TBHP (red) show increased staining with the CellROX® Deep Red Reagent as compared with cells pretreated with NAC before TBHP treatment (blue) and with untreated 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 min 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).|
Monitor live cells for lipid peroxidation in real time
The Image-iT® Lipid Peroxidation Kit provides a simple ratiometric method for detecting the oxidative degradation of cellular lipids in live cells (Figure 3). This kit supplies you with the hydrophobic BODIPY® 581⁄591 C11 reagent, which serves as a sensitive lipid peroxidation reporter, as well as cumene hydroperoxide for inducing lipid peroxidation. BODIPY® 581/591 C11 reagent localizes in cell membranes and exhibits a fluorescence emission shift from ~590 nm to ~510 nm upon oxidation by lipid hydroperoxides. The ratio of red fluorescence to green fluorescence provides a measure of lipid peroxidation that is independent of factors such as lipid density that may influence measurement with single-emission probes.
The Image-iT® Lipid Peroxidation Kit is easy to use—simply add the reagent (supplied ready-to-use in DMSO) to live cells in complete medium, incubate for 30 minutes, and measure the signal. This kit is compatible with fluorescence microscopy [5,6] including HCI, as well as with flow cytometry [7,8].
|Figure 3. Quantitation of lipid peroxidation in live cells using the Image-iT® Lipid Peroxidation Kit. U2OS cells were plated on 35 mm glass-bottom dishes (MatTek) and stained with 10 μM BODIPY® 581/591 C11 (provided in the Image-iT® Lipid Peroxidation Kit) for 30 min in complete growth medium at 37°C. Where specified, cells were pretreated with 150 μM α-tocopherol (a lipid-soluble antioxidant) for 30 min. Cells were then treated with DMSO (control), 100 μM menadione, 200 μM tert-butyl hydroperoxide (TBHP), or 200 μM cumene hydroperoxide (CH) for 2 hr at 37°C; all cells were stained with Hoechst® 33342 dye during the last 30 min of incubation. The cells were then washed three times with PBS and imaged on a Zeiss® Axiovert® inverted microscope using a 40x objective and appropriate optical filters. The signal intensity was quantitated at 510 nm and 590 nm using SlideBook™ 5.0 software. In control cells, low levels of lipid peroxidation are indicated by a relatively high 590/510 ratio, reflecting predominance of the reduced dye. After treatment with menadione, TBHP, and CH, the 590/510 ratios decreased dramatically, indicating significant oxidation of the dye and higher levels of lipid peroxidation; α-tocopherol pretreatment decreased lipid peroxidation in cells.|
Quantitate lipid peroxidation–derived protein modifications using versatile click chemistry
The Click-iT® Lipid Peroxidation Imaging Kit provides a click chemistry–based method to detect and quantitate lipid peroxidation–derived protein modifications in fixed cells (Figure 4). When incubated with live cells, Click-iT® LAA (linoleamide alkyne, included in the kit) incorporates into cellular membranes. Upon lipid peroxidation, the membrane-bound LAA is oxidized and produces 9- and 13-hydroperoxy-octadecadienoic acid (HPODE). These hydroperoxides decompose to α,β-unsaturated aldehydes that readily modify proteins surrounding them. Once cells are fixed, the resulting alkyne-containing proteins can be detected via a click chemistry reaction with Alexa Fluor® 488 azide (included in the kit).
Click-iT® LAA is also sold as a stand-alone reagent to allow detection of alkyne-containing proteins in fixed cells with any one of a number of azide-containing detection reagents. If the green-fluorescent Alexa Fluor® 488 azide is not compatible with your multiplex experiments, you can choose a longer-wavelength Alexa Fluor® azide, a tetramethylrhodamine azide, or a biotin azide for detection. Stained cells can be analyzed using traditional or quantitative fluorescence microscopy.
|Figure 4. Quantitation of lipid peroxidation in fixed cells using the Click-iT® Lipid Peroxidation Imaging Kit. Bovine pulmonary artery endothelial (BPAE) cells were plated on 35 mm glass-bottom dishes (MatTek) and incubated in complete growth medium at 37°C. For α-tocopherol pretreatment, cells were incubated with 150 μM α-tocopherol for 30 min. Cells were then treated with vehicle (DMSO) or 100 μM cumene hydroperoxide (CH), followed immediately by addition of 50 μM linoleamide alkyne (LAA, provided in the Click-iT® Lipid Peroxidation Imaging Kit). After a 2 hr incubation at 37°C, cells were fixed, permeabilized, and click-labeled with 5 μM Alexa Fluor® 488 azide (provided in the Click-iT® Lipid Peroxidation Imaging Kit) for 30 min and imaged on a Zeiss® Axiovert® inverted microscope. The signal intensity was quantitated using SlideBook™ 5.0 software; the intensity values were background-subtracted and plotted. The significant increase in signal intensity in CH-treated cells indicates elevated lipid peroxidation–derived protein modifications. α-Tocopherol pretreatment produced significant reduction of CH-induced protein modifications.|
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