The overall goal of this procedure is to isolate respiring mitochondria from cultured cells or mouse tissue, and then assess the strength of their membrane potential as a measure of cellular status. For cultured cells, this is accomplished by first plating and then treating the cells. Next, the cells are homogenized and the homogenate is passed through a needle to further release mitochondria from the cells.
For mouse tissue, the liver is excised from the mouse and then homogenized on ice with both types of starting material. Differential centrifugation is then used to isolate mitochondria from the rest of the cellular contents. After the mitochondria is subjected to some processing, including addition of the membrane potential sensitive fluorescent dye, TMRE fluorescence measurements are performed to calculate the strength of the membrane potential.
Ultimately, mitochondrial function and perturbations in mitochondrial membrane potential can be assessed using a membrane potential sensitive fluorescent dye in conjunction with a fluorescent plate reader When treated with common mitochondrial on couplers and inhibitors, Though this method can provide insight into cytokine induced apoptosis and mitochondria dysfunction in a LS.It can also be applied to any other system where comparison between two or more different samples of sought such as healthy versus disease or two different species of an animal with different metabolic rates. Visual demonstration of this method is critical. As the mitochondria isolation steps are difficult to learn.
This is because there are a lot of nuances when performing the technique that can be overlooked in traditional method sections. Begin this procedure with growing human embryonic kidney cells as described in the text protocol at 48 hours prior to cytokine treatment plate, 160, 000 cells into a T 1 75 flask to yield about 70%density at the time of treatment. Then serum starve the cells 24 hours later by aspirating the media and replacing it with the same volume of serum free media.
Finally, treat the serum starved cells by the addition of solubilized cytokines directly onto the cell culture media to perform isolation of mitochondria. First aspirate the media and wash the adherence cells twice with 15 milliliters of one x phosphate buffered saline or PBS. Each time following the wash, aspirate the buffer and scrape the flask to remove the adherent cells from the bottom.
Then add 15 milliliters of one XPBS to each flask, swirl and transfer to individual 15 milliliter conical tubes on ice. Once scraping is done, centrifuge the tubes for five minutes at 700 times G four degrees Celsius, using a tabletop centrifuge with a swinging bucket rotor following centrifugation, aspirate supernatants and resus. Suspend each pellet in one milliliter of mitochondrial isolation buffer.
Next, combine both suspensions and transfer to a small glass vessel for homogenization at the mitochondria resuspended and mitochondrial isolation buffer to the glass vessel until the solution reaches the first line With the pestle attached to a drill, proceed to homogenize the cells on ice at medium speed for three passes. Care should be taken to remove air bubbles when placing the pestle into the solution, not to remove the pestle above the liquid and to use a steady speed with continuous passes. Transfer the homogenized solution to a 50 milliliter conical tube.
Then draw the solution into a three milliliter syringe using an 18 gauge, one in one half inch needle and expel it back into the conical tube on ice with a 27 gauge one half inch needle. Take care to expel the solution against the inside wall of the tube to utilize that force for cell membrane disruption. Repeat the syringe steps a total of five times, then transfer the solution into a 15 milliliter conical tube and centrifuge for five minutes at 600 times G four degrees Celsius in a tabletop centrifuge using a swinging bucket rotor.
Mitochondria are in the supernatant after this first low speed spin, while some membranes and unbroken cells are pelleted, carefully remove the supernatant and distribute among three 1.5 milliliter tubes. Then centrifuge the three tubes in a fixed angle rotor at 10, 000 times G four degrees Celsius for five minutes. Mitochondria from cultured cells appear whitish in color.
Excise the liver from each animal and place an ice cold, one XPBS without calcium or magnesium to rinse away any blood. Transfer the liver tissue to a away dish on ice and chop it into fine pieces using a fresh razor blade for one minute. Next, add tissue and the appropriate buffer to the vessel until the buffer reaches the first line.
Proceed to homogenize the tissue on ice with the pestle by hand for five passes. Care should be taken to remove air bubbles when placing the pestle into the solution, not to remove the pestle above the liquid and to use a steady speed with continuous passes. Continue to process the samples with centrifugation and homogenization as solicit in the text protocol.
After combining the new supernatants with the first supernatant from each sample, centrifuge the tubes in a fixed angle rotor at 10, 000 times G four degrees Celsius for five minutes. Carefully aspirate the top white fluffy layer using a pipette tip and or pushing it to the sides of the tube. Then gently pour off the rest of the supernatant and resus.
Suspend the liver mitochondria pellets in 500 microliters of mitochondrial isolation. Buffer mitochondria from liver are brown. Quickly place the samples on ice immediately before reactions are set up.
Dilute the mitochondria to 0.5 milligrams per milliliter. Working concentration with experimental buffer. Add appropriate treatments as listed in the text protocol to separate tubes at one 10th, the total volume of diluted mitochondria.
Next, place the diluted mitochondria into each reaction tube and gently agitate each tube to ensure proper mixing. Incubate the reactions on the benchtop for seven minutes. Then add an equal volume of two micromolar tetraethyl, rod domine, ethyl ester, abbreviated TMRE to the mitochondrial reaction volume.
Again, gently agitating each tube to ensure proper mixing. After incubating the reactions on the benchtop for an additional seven minutes, pellet the mitochondria by centrifugation in a fixed angle rotor at 10, 000 times G four degrees Celsius for five minutes. Then load half of the supernatant volume of each sample onto a 3 84.
Well plate proceed to read the fluorescence of the samples and then calculate the fold difference in fluorescence between control and each treatment as described in the text protocol treatment of HEK 2 93 T cells with TNF alpha IL one beta and IFN gamma for 24 to 48 hours leads to progressive amounts of cell death. Cell viability was assessed using MTT assays that consistently demonstrate that there is an approximate 10%decrease in cell viability with 24 hour treatment and an approximate 20%decrease. With 48 hour treatment cells treated with similar concentrations yield similar cell death results at 48 hours.
And treatment with individual cytokines reveals that the decrease in viability is due to combinatorial effects. The viability data represent the mean standard deviation of three separate experiments. Each run in triplicate and the tightness of the error bars demonstrate the reproducibility of the data.
The percent decrease in membrane potential in mitochondria isolated from cytokine treated cells signifies that mitochondrial health was impacted by cytokine treatments, corroborating viability data Following this procedure. Other methods like assessment of a TP generation total a TP or oxygen consumption can be formed in order to assess additional questions like what energy and respiration differences among the different samples. After watching this video, you should have a good understanding of how to isolate intact functional mitochondria and assess the strength of their membrane potential as a reflection of the cellular status.
