This article details the methodology for assessing mitochondrial function in cerebral vascular endothelial (CVE) cells. The protocol involves culturing CVE cells, preparing an extracellular flux assay, and utilizing a bioanalyzer to measure mitochondrial activity.
Begin with cerebral vascular endothelial or CVE cells in culture media.
CVE cells are high energy-demanding cells that line the brain's blood vessels and are essential for brain physiology.
Place the cells into an extracellular flux cell culture plate. Incubate for cell attachment.
Replace the culture media with extracellular flux assay media that has the desired pH.
Incubate without carbon dioxide to maintain the pH.
Next, take an extracellular flux sensor cartridge, which includes sensors and a bottom plate.
Add a calibration solution to the plate and incubate without carbon dioxide to hydrate the sensors.
These sensors can measure the changes in oxygen concentration in the extracellular environment, indicative of mitochondrial activity.
Then, load the cartridge into a bioanalyzer machine and calibrate the sensors.
Replace the bottom plate with the plate containing the cells.
Measure the mitochondrial function of CVE cells.
After the appropriate number of subcultures, seed 1.6 x 104 cells in 80 microliters of complete medium per well onto a 96-well extracellular flux cell culture plate and place the plate in a 37 degrees Celsius incubator with 5% carbon dioxide.
The day before the mitochondrial functional assessment assay, hydrate an extracellular flux sensor cartridge with 150 microliters of extracellular flux calibrant solution, and incubate the plate overnight at 37 degrees Celsius without carbon dioxide.
The next morning, use the plate washer station to change the cell culture medium into pH 7.0 extracellular flux assay medium, then, incubate the cells at 37 degrees Celsius without carbon dioxide for 30 to 60 minutes. When the cells have equilibrated, load the hydrated cartridge into the bioanalyzer, and click the Start button to begin the calibration.
At the end of the calibration, click the Unlock Cartridge prompt to remove the cartridge bottom plate, and load the cell plate, then, open the data file to obtain the rate values from the bioanalyzer for export into the spreadsheet file.
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