Intracellular and Mitochondrial Reactive Oxygen Species Measurement in Primary Cultured Neurons

Abstract

Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules. ROS consist of radical oxygen species including superoxide anion (O₂ ^•-) and hydroxyl radical (•OH) and non-radical oxygen species such as hydrogen peroxide (H₂O₂), singlet oxygen (O₂). ROS are generated by mitochondrial oxidative phosphorylation, environmental stresses including UV or heat exposure, and cellular responses to xenobiotics ( Ray et al., 2012 ). Excessive ROS production over cellular antioxidant capacity induces oxidative stress which results in harmful effects such as cell and tissue damage. Sufficient evidence suggests that oxidative stresses are involved in cancers, cardiovascular disease, and neurodegenerative diseases including Alzheimer's disease and Parkinson disease (Waris and Ahsan, 2006). Though excessive level of ROS triggers detrimental effects, ROS also have been implicated to regulate cellular processes. Since ROS function is context dependent, measurement of ROS level is important to understand cellular processes (Finkel, 2011). This protocol describes how to detect intracellular and mitochondrial ROS in live cells using popular chemical fluorescent dyes.

Keywords: CM-H2DCFDA; Intracellular ROS; MitoSOX; Primary neuron; Reactive oxygen species (ROS).

Figure 1.. Equipment for the experiment.

A. Surgery instruments; B. Confocal microscope (LSM700) with live cell imaging system; C. Live cell chamber.

Figure 2.. The process of embryo extraction in pregnant Sprague Dawley rat (E17-E18)

(Steps 4-6)

Figure 3.. The process of extracting cortex region from the extracted whole brain

(Steps 7-9)

Figure 4.. The Process of making single cells of brain tissue and preparing for cell seeding

(Steps 10-17)

Figure 5.. Cell morphology at the 1st day (A) and after 8 days (B).

Scale bars, A = 500 μm; B = 200 μm.

Figure 6.. Detection of superoxide in rat primary neuronal cells’ mitochondria with MitoSOX^TM Red.

Oxidation of MitoSOX^TM Red reagent by superoxide produces red fluorescence. A. Bright field rat primary cell images; B. Red fluorescence generated by superoxide; C. Merged image. Scale bars = 50 μm.

Figure 7.. Measurement of ROS in intracellular compartment induced by Aβ in rat primary cells using CM-H₂DCFDA.

Green fluorescence represents intracellular ROS level of Aβ or vehicle treated sample. A. Vehicle-treated sample; B. 5 μM of Aβ treated sample. Scale bars = 50 μm.

Figure 8.. Flow chart of measuring method of ZEN black version of image measurement program

Figure 9.. The tab to control 'speed' and 'averaging' in ZEN black version

Figure 10.. Analysis flowchart of ZEN blue version.

Run Zen blue version, an analysis tool provided by Zeiss confocal equipment. Open the image you want to analyze and click the ‘Analysis’ tab. Press the ‘Setup Image Analysis’ button to set the analysis method (see Figure 10-1). Enter the proper analytical condition in order. In particular, set the appropriate threshold value (When you click on the area where CM-H₂DCFDA emits fluorescence, the default value is automatically set) in the 3rd step (The analysis setting value must be set up based on the positive control. The settings of all images to be analyzed should be applied equally). Identify the area you want to measure as shown in Figure 10-4. Set the results (Fluorescence mean, Standard deviation, Fluorescence dot number, etc.) you want to obtain and press the ‘Finish’ button. Finally, check the value by pressing the ‘Analysis’ button.

Figure 11.. Analysis flowchart of ImageJ.

To measure the intensity of fluorescence using ImageJ, several preliminary steps are required. After opening the image you want to analyze, you have to separate the fluorescent area that you want to measure (In flowchart 3, set the range to be measured while adjusting the threshold value. see Figure 11-2, 11-3). Click on the 'Set Measurements' tab as shown in flowchart 4 to set the results you want to obtain. Click the 'Measure' tab and acquire the results.