The overall goal of this methodology is to establish the use of genetically-encoded redox sensors for the specific assessment of toxicant-induced perturbations in redox homeostasis. By overcoming the significant limitations of conventional approaches used with oxidative stress, this method will help answer key questions pertaining to toxicological oxidative stress. The main advantage of this technique is that it combines a high degree of specificity and sensitivity with unparalleled spacio-temporal resolution to investigate toxicological oxidative stress in living cells.

Perform all imaging analysis using a confocal microscope equipped with laser lines at 404 and 488 nanometers, and environmental controls to maintain an appropriate temperature, humidity, and/or gas concentration suitable for the cells throughout the duration of the experiment. If using high values of relative humidity, use an objective heater and/or heating tape to keep any surfaces that may come in contact with the humidified atmosphere at or slightly above the temperature at which the humidity is generated to prevent condensation. Turn on all microscope components, and set up all equipment required for sequential excitation at 488 and 404 nanometers with emission at 510 nanometers.

Ensure all components of the optical configuration are set appropriately for real-time acquisition. Set up a stage-top environmental chamber to maintain constant temperature at 37 degrees celsius, 5%CO2 atmosphere, and greater than 95%humidity. Prior to starting image acquisition, equilibrate the environmental chamber for at least 10 minutes after the initial set-up of all environmental controls.

Place the dish of cells on the stage top within the environmental chamber. With the desired objective lens, find the focal plane of cells using the eyepiece and white light and ensure normal morphology. A 1.4 numerical aperture 60 X violet corrected oil-immersed objective lens is commonly used, which permits identification of intracellular compartments while maximizing the optical resolution of the confocal system.

Check the fluorescence expression of the cells in the field of view while visualizing the cells under wide-field fluorescence illumination with an appropriate filter set such as flourescian-isothioanate or FITC. At this point, using wide-field illumination while looking through the eyepiece is more convenient because it is easier to move the dish to select a field of cells to study. Once a desired field of view is found, close the environmental chamber.

Use the focus maintaining feature to facilitate maintenance of a stable focal plane throughout the study. Next, set up acquisition parameters to ensure optimal assessment of the sensor of interest across the desired exposure period. To do so, adjust the laser power for excitation at 488 nanometers and emission at 510 nanometers.

Choose a laser power level that allows visualization of the cells and keep this constant between samples or replicate dishes. Use the confocal controls of the acquisition software to ensure that the selected focal plane has been optimized for maximal florescence emission intensity in the center of the cells by scanning at 488 nanometers while adjusting the Z-plane. Use a high gain setting while adjusting for the Z-plane with the most over-exposed cells.

Once the appropriate focal plane has been found, return the gain to a setting that is most optimal for the fluorescence of the reporter being used without over-saturating the pixels being observed. Then, use the gain to fine-tune the baseline fluorescence. With roGFP2, establish the baseline near the upper limit of the instrument without over-saturation, as these cells will lose 510 nanometer fluorescence induced by 488 nanometer excitation when the glutathione redox potential increases.

Repeat these steps with excitation at 404 nanometers and emission at 510 nanometers. Gain settings for the 404 nanometer excitation wavelength are opposite to those used with 488 nanometer excitation for each sensor. In general, fluorescence of the 404 nanometer excitation will be considerably lower than that obtainable with the 488 nanometer excitation in cells expressing roGFP2, as a 404 nanometer peek is a relatively minor excitation maximum for this sensor.

Set up the acquisition software to sequentially excite the two excitation wavelengths and collect emissions for both at 510 nanometers and at a predetermined time interval throughout the desired length of the experiment. For real-time assessment of experimental parameters, choose at least five to 10 sensor-expressing cells in the field, and establish them as regions of interest to monitor their fluorescence changes during the experiment. Next, dissolve the environmental toxicant, 9, 10-PQ in dimethyl sulfoxide to a concentration of 15 millimolar.

Dilute the dissolved toxicant in basal-cell media to a 250 micromolar working solution for a final concentration of 25 micromolar. Additionally, prepare a working solution of hydrogen peroxide for later use in this protocol. Once the experimental parameters have been defined, begin the time course acquisition.

Establish a baseline period of at least five minutes prior to starting xenobiotic exposures. Expose the cells to the toxicant being investigated using conventional approaches for in-vitro dosing. Monitor changes in fluorescence during the exposure period.

Perform subsequent injections as needed. Take great care not to shift the dish during injections so that the same cells are followed throughout the entire time course while imaged in the same focal plane. At the end of the experiment, expose the cells to the appropriate controls to fully oxidize and reduce roGFP2.

The addition of control simuli validates sensor responsiveness at the end of the exposure and confirms that the responses observed are not the direct result of damage to the forport. So, it is especially important in the initial evaluation of a toxin. For these studies, use one millimolar hydrogen peroxide to determine the maximum sensor response.

Wait at least five minutes to allow the sensor to respond and then inject a reducing agent such as DTT to reduce the sensor and return it to a level of fluorescence at or near its established baseline. Finally, perform data analysis as described in the text protocol. Shown here are representative results illustrating an established dose response generated using the bronchial epithelial cell line Bees 2B expressing roGFP2.

Cursor shown to exposure five micromolar, 25 micromolar, or 100 micromolar doses of hydrogen peroxide. Exposure of bees 2B cells to a previously untested environmental toxicant, 9, 10-PQ induces a gradual but potent increase in the intracellular glutathione redox potential as assessed by roGFP2. Here, each line depicts the raw roGFP2 ratio of individual cells in the dish.

Although the test compound produces a maximal response comparable to one millimolar hydrogen peroxide, this known redox cycler caused a change in the sensor that was fully reduced with the addition of five millimolar DTT, suggesting that the sensor remains functional following exposure to this redox-active organic compound. This approach will aid toxicologists in investigating key mechanistic events in toxican-induced oxidative stress, paving the way for more focus studies in the emergent field of redox toxicology across a broad spectrum of toxicants and cell types. After watching this video, you should have a good understanding of how to use genetically encoded sensors and live-cell microscopy to monitor pivotal markers of toxicological oxidative stress with high sensitivity and specificity at unmatched spacio-temporal resolution.