High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Our lab studies the impact of environmental pollutants, like heavy metals on liver function and diseases related to reactive oxygen species production. This protocol aims to quantitatively measure ROS levels in cells using a dihydroethidium fluorescence probe. This protocol uses a high-content screening platform to quantitatively measure intracellular ROS species levels in hepatocellular lines.

Measuring intracellular ROS level accurately is of critical importance in high-throughput toxicology study, which is a key area of interest in our lab. DHE fluorescence is a highly specific method for measuring total ROS levels, particularly targeting the superoxide radical, and giving it a significant advantage over other commonly-used ROS fluorescence probe. Additionally, this protocol offer the unique ability to measure total cellular ROS level in a high-throughput platform.

Our protocol is a robust and reproducible choice to characterize intracellular total ROS production. High-throughput approaches to measure ROS levels are helpful in a variety of studies, such as toxicology, drug screening, and cancer biology, which will be our focus in the future. To begin, seed 200 microliters volume of 10, 000 hepatocellular carcinoma cells into a 96-well plate with DMEM and 10%FBS.

Incubate the cells overnight at 37 degrees Celsius in a humidified incubator under 5%carbon dioxide. The next day, replace the culture media and treat the cells with varying concentrations of hydrogen peroxide and menadione. Dissolve five milligrams of DHE in one milliliter of DMSO.

For the working solution, dilute a 100 micromoles aliquot with prewarmed DMEM media to a concentration of 10 micromoles. Vortex the working dye solution for five to 10 seconds to ensure proper mixing. Now, remove the test media from each well before staining the carcinoma cells.

Then, gently wash each well with PBS. Pipette 100 microliters of the working dye solution into each well. Incubate the plate at 37 degrees Celsius for 30 minutes.

Remove the DHE-containing media from each well. Use a multichannel pipette to wash each well three times with PBS. Now, pipette 200 microliters of PBS containing Hoechst 33342 nuclear staining dye into each well.

After incubation, transfer the plate to a high-content screening platform. Launch the Cellomics CX7 high-content screening software. Calibrate the imaging platform as per the manufacturer's specifications.

Open the system parameters specific to the plate brand in the instrument database. Next, carefully place the plate with the samples onto the heated stage of the imaging system. Ensure that the plate is securely positioned and in the correct orientation.

Then, gently press down on the microplate to ensure it rests flat against the stage. Once the plate is properly positioned, press and hold the Control key. Then, click on Control and okay to open the plate load/unload dialogue box.

To select the imaging parameters, open the Target Activation mode in the Cellomics BioApplications interface. Create a new protocol for dual-channel data acquisition compatible with nuclear staining and DHE fluorescence probe. Next, specify the required objectives for image acquisition, then choose the appropriate magnification.

Now, specify the exposure time, camera binning, and Z-stack interval. Once the imaging parameters are set, identify the primary tracking object of interest using the different algorithms in the instrument. After segmenting the identified object, validate the primary object based on the specific size, shape, and intensity parameters unique to each cell type.

Define the region of interest around the object and set a 20-micrometer circle around it. Click on the Population Characterization tab, then set the scan limit to 1, 000 cells per well with a minimum of 10 objects per field. Now, click on Launch View to scan each well plate.

Then, launch the view analysis software and export the quantitative data parameters in a csv format for additional analysis. Peroxide exposure resulted in a statistically significant increment of the ROS-induced fluorescence across all the cell lines in a dose-dependent manner. With menadione, the JHH-4 cells showed a dose-dependent increment in fluorescent intensity.

However, the significant difference in fluorescence was observed only at higher concentrations in the other two cell lines.