Mitochondrial respiration is a principle generator of cellular energy under most circumstances, and an important indicator of cellular metabolism and fitness. Thus, mitochondrial functions delineate the bioenergetic cellular state. This protocol describes a technique for quantifying mitochondrial respiration in G living cells as a bioenergetic parameter.
The technique described in this protocol help us define the bioenergetic mechanism of some polyphenols, like resveratrol and coircitene. It also serve as a tool to measure the pentose metabolism, also of PTSPTs and explained bioenergetic changing behind the cab tree and ground work effects. We are interested in deciphering how cells acquire some phenotypes, like cab tree and ground work effects, with significant changes in cellular bioenergetics.
Mitochondrial respiration is an essential tool for elucidating the molecular mechanisms behind this metabolic phenomena. To begin, inoculate a 10 milliliter, glass test tube containing three milliliters of YPD medium with 250 microliters of glycerol preserved yeast cells. Incubate the test tube overnight at 30 degrees Celsius with constant agitation at 200 revolutions per minute.
Then, using a sterile loop, streak a YPD auger plate with the yeast cells grown in the 10 milliliter glass test tube. Incubate the Petri dish at 30 degrees Celsius until isolated colonies appear. To prepare a pre-inoculum, inoculate three milliliters of YPD medium at 2%glucose with two or three isolated yeast colonies and incubate.
Then obtain 100 milliliters of sterile synthetic complete medium in a 500 milliliter Erlenmeyer flask. Inoculate the media with the overnight pre-inoculum yeast culture at an initial optical density of 0.1 at 600 nanometers. Add the chemical challenge such as 100 micromolar resveratrol to test mitochondrial respiration.
Next, pour the mid log phase cells into an empty, pre-weighed 15 milliliter conical tube, and centrifuge the cells at 4, 000 G for five minutes. After discarding the supernatant, wash the pellet three times with 25 milliliters of deionized water. Weigh the conical tube containing the washed pellet to obtain the wet weight by subtracting the empty tube's weight.
Then re-suspend the washed pellet in two milliliters of deionized water. To begin, obtain the yeast cells treated with appropriate drugs, such as resveratrol. Place five milliliters of 10 millimolar MES-TEA buffer and 50 milligrams of wet cells into the polar graft chamber.
Gently, position the Clark type oxygen electrode, ensuring no bubbles are formed. Turn on the YSI 5300A monitor and the data acquisition computer. Select channel one setting dial to air, and adjust channel one to 100%with the channel one calibration dial.
Start recording the data and begin measuring time using a chronometer. Using a chromatography syringe, add the oxidizable substrate to a final concentration of 10 millimolar, and keep the chamber closed with constant agitation. Then, employing a chromatography syringe, add oligo mycin to obtain a final concentration of 0.01 millimolar in the oximeter chamber and record the air percentage consumption.
Similarly, add CCCP to a final concentration of 0.015 millimolar in the oximeter chamber to determine maximal respiration capacity. Next, add electron transport chain inhibitors, tooth and oil triflurositone, or TTFA, at one millimolar, followed by antimycin A at one milligram per milliliter. Wash the chamber three times each with 70%ethanol, followed by deionized water, before proceeding to the next carbon source.
To begin, perform an oxygen consumption assay with yeast cells, using various carbon sources and controls. Create a new project file in the statistical software and choose XY in the create section. Then select enter, or import data into a new table in the data table section.
In the options section, select numbers in the X subsection and in the Y subsection. Click on enter three replicate values in side-by-side sub columns. Enter time data in the X columns and input the air percentage data in the Y columns.
To perform a linear regression analysis, click analyze in the menu bar and under analysis, select simple linear regression on XY analysis, then click okay. Obtain the slope value from the results section in data tables. Copy the calculated slope values into a data sheet.
Subtract the slope value obtained from the respiratory inhibitors mix Mix from those of the oxidizable substrate, oligomycin, and CCCP to discard other oxygen consumption sources. Now, multiply the slope values by 237 micromolar of oxygen, which represents the solubility of oxygen in that condition, and buffer. Convert the values to micromolar of oxygen per minute, adjusting according to how time is per plotted.
Divide the results by 50, representing the milligrams of cells used in the experiment. Finally, the oxygen consumption is expressed as micromolar of oxygen per milligram of cells per minute. Cells grown in 0.5%glucose showed significantly higher oxygen consumption in basal respiration, ATP linked respiration, and maximal respiratory capacity, compared to those grown in 5%glucose.
Quercetin significantly reduced mitochondrial respiration in cells grown with 0.5%glucose, affecting basal, ATP linked, and maximal respiration. On the other hand, quercetin did not, significantly, affect mitochondrial respiration in cells, grown in 5%glucose.
