Examining Oxidative Stress using the Tali® Image-Based Cytometer and CellROX® Orange Reagent
Generation of reactive oxygen species (ROS) is an expected product of aerobic metabolism. At low levels, these species can serve as signaling molecules. However, higher concentrations of these reactive molecules can damage cellular proteins, lipids, and DNA. Consequently, there are multiple mechanisms in place to neutralize ROS, including glutathione and enzymes such as superoxide dismutase and catalase. Despite these protective mechanisms, imbalances in ROS can occur. Elevated levels of ROS are associated with several diseases, including cancer. Increased levels of ROS can also indicate prelethal toxicity induced by various drugs. Accordingly, the ability to measure oxidative stress is an important aspect of toxicological profiling and an assessment of cell health
In this application note, we demonstrate the capability of the Tali® Image-Based Cytometer to assess oxidative stress in cells. CellROX® Orange Reagent, a new probe for ROS detection in live cells, is a cellpermeant dye that is nonfluorescent until oxidized by ROS. CellROX® Orange Reagent has excitation/emission maxima of 545/565 nm, making it compatible with the RFP channel of the Tali® instrument, which is excited at 530 nm and uses a 580 nm longpass emission filter. Cells stained with CellROX® Orange Reagent can be analyzed using the Tali® Image-Based Cytometer to assess increases in ROS levels.
Materials and Methods
Materials and methods Menadione and tert-butyl hydroperoxide (tBHP) were used to induce oxidative stress in Jurkat cells. Cells were treated with 200 μM tBHP for 1 hour, 100 μM menadione for 2 hours, or a vehicle control. After drug treatment, CellROX® Orange Reagent was directly added to 1 mL of cells (at 1 x 106 cells/mL) in complete medium at a 1:500 dilution (2 μL dye to 1 mL cell solution). Samples were incubated for 30 minutes at 37°C, centrifuged once to remove medium and excess dye, and then resuspended in Hank's balanced salt solution.
After labeling with CellROX® Orange Reagent, cells were analyzed with the Tali® Image-Based Cytometer using the RFP channel, collecting 9 fields per sample. In this assay, “RFP fluorescence” represented the fluorescence signal from CellROX® Orange Reagent. Untreated cells, which were also labeled with CellROX® Orange Reagent, were used to determine baseline levels of oxidative activity and to set the fluorescence threshold for the Tali® instrument.
Because the Tali® instrument allows users to visualize cells, an accurate threshold can be set and confirmed. This threshold was set manually and confirmed visually, and all cells with signal greater than the threshold (represented by the blue vertical line) were counted by the Tali® instrument as positive.
Results and Conclusion
In this experiment, there was a greater response with tBHP treatment than with menadione. This is expected, because these two agents likely produce different profiles of ROS within cells. Treatment with tBHP produced an increase in ROS levels above control in 95% of the cells (Figure 1). In contrast, only 17% of the cells treated with menadione showed an increase in ROS above control (Figure 2). Using the Tali® Image-Based Cytometer, the percentage of cells with increased ROS levels was easily evaluated. CellROX® Orange Reagent is a novel probe for evaluating oxidative stress in live cells. Unlike traditional fluorescein-based probes for ROS, CellROX® Orange Reagent can be loaded in complete serum-containing media and requires minimal dilution and wash steps. Obtaining quantitative data using traditional fluorescence imaging is typically laborious, and automated high-content screening methods can require expensive instrumentation. By staining cells with CellROX® Orange Reagent and using the Tali® Image-Based Cytometer to visualize, analyze, and quantify results, a reliable measurement of oxidatively stressed cells in a population was quickly and easily obtained.
Figure 1. Measuring oxidative stress in cells treated with tBHP using the Tali® cytometer. Jurkat cells were treated with vehicle control (A) or with 200 μM tert-butyl hydroperoxide (tBHP) (B) for 1 hour. Cells were stained with CellROX® Orange Reagent and then analyzed with the Tali® Image-Based Cytometer using the RFP channel, collecting 9 fields per sample.
Figure 2. Measuring oxidative stress in cells treated with menadione using the Tali® cytometer. Jurkat cells were treated with vehicle control (A) or with 100 μM menadione (B) for 2 hours. Cells were then stained with CellROX® Orange Reagent and analyzed on the Tali® Image-Based Cytometer using the RFP channel, collecting 9 fields per sample.