The goal of this procedure is to determine the mitochondrial membrane potential and levels of reactive oxygen species in primary cortical neurons using the fluorescent probes, TMRM, and H 2D CFD respectively. This is accomplished by first preparing stock and working concentrations of the TMRM and H 2D CFDA. The second step is to incubate the cortical neurons with TMRM and H 2D CFDA at room temperature for 45 minutes in Thai roads buffer.
The third step is to image the fluorescence intensity of TMRM and DCF in live cortical neurons using 5 15 5 17 nanometer and 4 88 5 15 nanometer excitation emission respectively. The changes in TMR in fluorescence will be monitored in the presence of the mitochondrial unco FCCP, while the changes in DCF fluorescence will be monitored in the presence of hydrogen peroxide. The final step of this procedure is the collection, normalization, and analysis of data.
Ultimately, results are obtained by plotting a graph that shows the relative levels of TMRM and DCF fluorescence intensity before and after FCCP and hydrogen peroxide treatment respectively. Hello, I'm Joanna Koska. Welcome to my lab at Loyola University Chicago.
Today we'll show you how to measure mitochondrial membrane potential and reactive oxygen species in rat cortical neurons. The procedure will be demonstrated by dhi. Hi, I'm de Denise Jossi.
Using this procedure, mitochondrial membrane potential can be measured at single mitochondrial label and reactive oxygen species at single cell label. So let's get started. Prepare a 10 millimolar stock of TMRM by dissolving five milligrams of TMRM in one milliliter of anhydrous dimethyl sulf oxide vortex for one minute.
Then make aliquots store the aliquots at minus 20 degrees Celsius. Protect from light and use within one month. Next, prepare a 10 millimolar stock of H 2D CFDA by dissolving 4.87 milligrams of H 2D CFDA in one milliliter of anhydrous dimethyl sulf oxide.
Similarly, vortex for one minute, then make aliquots and store them at minus 20 degrees Celsius. Protect from light and use within one week to load the rat cortical neurons with TMRM first. Wash the cultured neurons three times with tyro's buffer.
Then prepare 20 nano molar TMRM by diluting the 10 millimolar TMRM stock 1000 times in tb. Then add two microliters of the diluted TMRM per one milliliter of tb. Incubate the neurons with TMRM for 45 minutes in the dark at room temperature.
After 45 minutes, mount the culture dish on the stage of the microscope and start imaging to load the rat cortical neurons with H 2D CFDA, wash the cultured neurons three times with tb. Next, prepare two micromolar of H 2D CFDA by diluting the 10 millimolar H 2D CFDA stock 10 times in tb. And then add two microliters of diluted H 2D CFDA per one milliliter of tb.
Then incubate the neurons with H 2D CFDA for 45 minutes in the dark at room temperature after 45 minutes. Wash the neurons four times with TB to remove excess fluorescent indicator before obtaining images to perform live imaging of neurons incubated with TMRM confocal laser scanning microscopy with the application of live time series program is used. Apply low resolution and attenuated laser power to minimize the time needed to obtain images and to avoid photo bleaching.
Next, adjust the focus of the mounted neurons loaded with TMRM using reflected light. Examine the TMRM fluorescence by illumination at five 14 nanometers and detection at five 70 nanometers. Set the detection gain of the camera just below the saturation level once all parameters, which include resolution, laser power detection, gain of the camera and time-lapse interval to obtain images are set.
Do not change these settings between experiments. Next, change the field. Start collecting images to test changes in delta PSY M stimulus, such as one micromolar of FCCP or two microgram per milliliter of oligo mycin can be applied, which will significantly depolarize or hyperpolarize the mitochondrial membrane potential respectively.
These changes will be reflected by a decrease in TMRM fluorescence intensity compared with the baseline fluorescence intensity in the case of FCCP or an increase in TMRM fluorescence intensity. In the case of a LIGA myin sin to perform live imaging of neurons incubated with H 2D CFDA first mount the culture dish on the stage of a microscope, adjust the focus of the cells using reflected light. Examine the DCF fluorescence by excitation at 4 88 nanometers and emission at five 15 nanometers.
Next, adjust the laser power to five to 7%detect a gain and resolution of 2 56 by 2 56. Do not change these settings between experiments. Then set the frequency for obtaining live images.
Using the Time series program, select a new field and start acquiring images to detect changes in R os levels. Treat cells with 100 to 200 micromolar of hydrogen peroxide. This will be reflected by an increase in DCF fluorescence intensity compared with baseline level.
Use the region of interest tool from the LSM program to select the areas. Then select ROIs from mitochondrial regions or ROIs from the entire cell body in image cells. To measure the fluorescence intensities of TMRM or ROS respectively.
Select regions next to the cells. To calculate the background fluorescence intensity, take several measurements and to calculate the average background intensity, subtract the average background fluorescence intensity from average fluorescence intensities of regions of interest in each cell for each time point using Microsoft Excel. After subtracting background intensity, normalize the TMRM or DCF fluorescence intensity to the baseline fluorescence.
Using this formula, then use the Sigma plot program to generate the plot showing the changes in fluorescence intensity over time. Here is an example of a fluorescence image of rat cortical neurons incubated with TMRM. The addition of F-C-C-P-A mitochondrial unco leads to mitochondrial depolarization and a loss of TMRM fluorescence intensity.
The baseline TMRM fluorescence level remains stable before the addition of FCCP. Quantitative analysis of TMRM fluorescence changes over time shows a significant decrease in TMRM fluorescence after addition of FCCP. Here's an example of a fluorescence image of rat cortical neurons loaded with DCF.
The baseline DCF fluorescence level remains unchanged in the first 120 seconds before application of hydrogen peroxide. The addition of hydrogen peroxide results in an increase in DCF fluorescence intensity in the cell bodies. Time-lapse measurements of DCF fluorescence show its steady levels which have increased after hydrogen peroxide treatments.
In this procedure, it is important to remember to use a low laser power and fast scan speed to avoid the artifacts from photo bleaching and phototoxicity. Have a good luck with your experiment.
