The overall goal of this procedure is to use high-resolution respirometry to determine mitochondrial oxygen consumption. This method can help answer key questions in the syndromes and diseases associated with mitochondrial dysfunction such as;sepsis, diverse neurological diseases and age-related disorders. The main advantages of this technique is that it has higher sensitivity and the ability to perform substrate on coupled inhibitor titrations experiments with a small number of biological samples such as intact or permeabilized cells.
Demonstrating the procedure will be Sandra Nansoz, a technician from my laboratory. Before beginning the procedure, air calibrate the polarographic oxygen sensors then resuspend the cells in respiration buffer to a one times ten to the sixth cells per milliliter concentration and replace the respiration medium in one chamber of the oxygraph with 2.1 milliliters of the cell suspension. Close the chamber with the stopper and set the magnetic stirring bar in the chamber to 700 rotations per minute.
Then record the cellular respiration for 5 to 10 minutes until a stable oxygen flux signal is achieved. Next, use a syringe to inject two microliters of Rotenone through the titanium injection port into the oxygraph chamber and record the cellular respiration for another 5 to 10 minutes. When a stable oxygen flux signal is achieved, inject 20 microliters of one molar succinate followed by 10 microliters of 0.5 molar ADP.
Then inject two volumes of two microliters of two millimolar digitonin and record the cellular respiration for two to five minutes after each injection followed by the step-wise injection of two to four microliters of two millimolar digitonin until the oxygen flux signal reaches the maximum level and further digitonin injections do not increase the respiration rate. For evaluation of the mitochondrial outer membrane integrity prepare an oxygraph chamber as just demonstrated and inject two microliters of eight millimolar digitonin to permeabilize the cells. After five minutes, inject 20 microliters of one molar succinate and record the cellular respiration for 5 to 10 minutes until a stable oxygen flux signal is achieved.
Then inject 10 microliters of 0.5 molar ADP to stimulate complex two, and to induce an oxygen consumption increase. When a stable flux signal is achieved, inject five microliters of four millimolar cytochrome c followed by one microliter of four milligrams per milliliter of ligamycin. To measure the ADP-stimulated respiration of the liver hepatocellular carcinoma cells, prepare an oygraph chamber as just demonstrated, and inject two microliters of eight millimolar digitonin into the oxygen chamber to permeabilize the cells for five minutes, followed by the injection of 12.5 microliters of 0.8 molar mallade and 10 microliters of two molar glutamate.
When a stable oxygen flux is achieved, inject 10 microliters of 0.5 molar ADP to increase the oxygen consumption, followed by two microliters of 0.2 millimolar rotenone and 20 microliters of one molar succinate. Next, inject two microliters of five millimolar antimycin and record the cellular respiration. When the signal decreases and stabilizes, inject 2.5 microliters of 0.8 millimolar escorbate followed by the immediate injection of 2.5 microliters of 0.2 millimolar TMPD, recording the cellular respiration until the oxygen flux signal increases and stabilizes.
Then inject 10 microliters of one molar sodium azite into the oxygraph chamber, and record the cellular aspiration until the oxygen flux signal decreases and stabilizes. To measure the intact cell oxygen consumption, prepare an oxygraph chamber as just demonstrated and inject 1 microliter of four milligrams per milliliter aligimicyn followed by one and three microliter injections of 0.2 millimolar FCCP. Next, titrate the FCCP in 0.1 to 0.3 micromolar steps by injecting one to three microliters of 0.2 to one millimolar FCCP until the oxygen flux signal reaches its maximal level with no further increases, and then begins to decline.
Then, inject two microliters of 0.2 millimolar rotenone and two microliters of five millimolar antimycin A and record the respiration until the oxygen flux signal decreases and stabilizes. In the absence of digitonin, cellular respiration is very low, and the respiration of intact, non-permeabilized cells is not stimulated in the presence of mitochondrial substrate and ADP. Upon stepwise addition of digitonin, however, the cellular plasma membrane becomes permeabilized and the mitochondrial respiration increases until full permeabilization, at which time the succinate and ADP enter the cells.
Mitochondrial outer membrane integrity can be compromised, however, if excessive amounts of digitonin are employed, inducing a reduction in complex to dependent state three respiration. Cytochrome c does not enhance complex to dependent state three respiration in digitonin-treated cells, indicating that there is no loss of cytochrome c from the mitochondrial outer membrane, and that mitochondrial integrity is preserved, even though cytochrome c can enhance the respiration of cells treated with very high doses of digitonin. The addition of exogenous substrates of mitochondrial complex activity post-digitonin permeablization induces an increase in mitochondrial respiratory chain complexes one, two, and four maximal respiratory rates.
The generation of reduced respiratory levels may be due to contamination of the oxygraph chambers with mitochondrial inhibitors from the previous experiment. In the presence of alygimycin and the sequential addition of FCCP, the maximal uncoupled respiratory rate of the cells is observed. Once mastered, any of these techniques can be done in less than one hour if it is performed properly.
While attempting this procedure, it is very important to remember wash oxygraph chambers and stoppers extensively after each experiment. Following this procedure, other methods like measurements of cellular ATP content, each presentate in cymatic activity, mitochondrial substrate levels can be performed in order to answer additional questions like whether changes in respiratory rates are due to lack of mitochondrial substrates or reduced ATP cyntates activity. After its development, this technique paved the way for researchers in the field of bionegetics to explore mitochondrial function in fields such as acquired and genetic mitochondrial diseases, oxydative stress, skin perfusion injury and aging in intact or permeabilized cells or fibers and isolated mitochondria.
After watching this video, you should have a good understanding of how to assist mitochondria function in permeabilized and intact cells using high-resolution respirometry. Don't forget that working with antimycin a, rotenone, sodium iozide, FCCP and aricomycin can be extremely dangerous and precautions such as wearing gloves and laboratory coats should always be taken while performing this procedure.
