Mitochondrial function is critical to cellular homeostasis and oxygen consumption rate is a decisive indicator of mitochondrial health. This protocol provides detailed instructions towards analyzing oxygen consumption rate in C.elegans. This protocol makes use of live, mobile C.elegans providing a legal measurement of mitochondrial function.
Elevating the need to isolate mitochondria, a step that could potentially effect it's integrity. To begin, transfer L4 larvae of desired genetic backgrounds onto a freshly seeded lawn of Escherichia coli on nematode growth media or NGM plates. Use at least two 100 mm or three 60 mm plates for each strain.
Incubate the worms at the appropriate growth temperature for three to four days, or until plates are concentrated with a large number of eggs and gravid worms. Wash the eggs and worms off the plates using approximately 6 milliliters of M9 buffer per 100 millimeter plate of nematodes. Using glass pasture pipettes, transfer the eggs and worms into individual 15 milliliter centrifuge tubes for each strain.
Spin theses tubes down for three minutes at 6, 180 g. Following centrifugation, aspirate out the M9 buffer retaining just the animal and egg pellet. Add three to four milliliters of bleach solution to each tube and intermittently vortex for six minutes.
Then, add M9 buffer to fill each tube and spin at 6, 180 g for one minute. Aspirate the supernatant and repeat this wash with the M9, three times. Following the wash, move the egg pellet to a fresh 15 milliliter tube containing approximately nine milliliters of M9 buffer.
Synchronize the freshly hatched worms by nutating at 20 degrees Celsius for 16 to 48 hours. After spinning these tubes down at 6, 180 g for 1.5 minutes, put the synchronized L-1 animals down on a freshly seeded lawn of OP50 on individual NGM plates. Keep the plates at 20 degrees Celsius until the animals reach the L-4 larval stage after approximately 42 hours.
At this time, use a platinum pick to move the L4 larvae to OP50 seeded NGM plates containing 0.5 milligrams per milliliter of FUdR to prevent the from producing progeny. Hydrate the sensor cartridge by adding 200 microliters of distilled water to each well on the plate, ensuring that the sensor probes are submerged. Store the plates overnight at room temperature.
Turn off the heating element within the respirometer interface. Storing the machine inside a 15 degree Celsius incubator lowers the core temperature within the machine and prevents the animals from overheating during the assay. After overnight incubation, pipette out and discard the distilled water used to hydrate the sensor probes, and replace it with 200 microliters of the calibrant solution in each well.
Dilute the FCCP stock solution to 100 micromolar FCCP in distilled water. Then, add 20 microliters of the diluted solution to injection port A in the sensor cartridge. Add 22 microliters of 400 millimolar sodium azides to injection port B of the sensor cartridge.
Now, turn on the respirometer. On the home screen, select start and the template pages will appear. On the templates page, select blank"or a previously designed template.
The groups page will appear. On the groups page, select wells A and H as the background wells, and assign the remaining six wells into appropriate groups according to the experimental plan. Access the protocol page by clicking the arrow on the lower right, and ensure that the equilibrate, basal, and injections one and two button are selected.
Adjust the number of readings of basal OCR as well as maximum OCR and non-mitochondrial OCR. Select the arrow on the lower right corner. A prompt to load the sensor cartridge plate will appear.
Ensure that the sensor cartridge plate is loaded in the right orientation, following the instructions on the reminder prompt screen. The respirameter will now equilibrate the cartridge. Add 200 microliters of M9 buffer into each of the eight wells and surrounding reservoirs of the cell plate.
Pick approximately 100 worms from each strain onto unseeded NGM plates, and allow to rest for two to three minutes. Then, wet the end of the platinum pick with M9 buffer and pick 20 age synchronized animals into wells B through G, leaving the background wells A and H empty. Now, click OK on the respirometer software to eject the plate containing calibration buffer.
Remove the plate from the respirometer leaving the sensor cartridge inside the instrument. Replace the calibrant plate with the plate containing the animals. Load plates and close the door by hitting continue and allow the assay to run.
Once the assay run is complete, follow the prompts on the screen to remove the cell plate and sensor cartridge. Insert a flash drive into the USB port to save the run data war format. Remove the sensor cartridge and allow the animals to settle to the bottom of the wells for approximately two minutes.
Place the cell plates under a stereo dissecting microscope and count the number of animals per well. Open the analysis software, followed by the normalization tab, to normalize oxygen consumption rates to animal number. Apply appropriate levels for the various groups under the modify"tab, and export the file as a prism file for further analysis.
The average of the first five measurements is the basal OCR. The average of the five measurements after FCCP addition is the maximal OCR. Finally, the average of the last five measurements after sodium azide addition is the non-mitochondrial respiration rate.
Since calcium dysregulation can result in altered mitochondrial function, OCR in both the sel-12 mutants and wild type animals were measured to examine the effect of sel-12 mutations on mitochondrial function and health. Wild type animals consistently showed basal OCR rates below five picomoles per minute, per worm. While all three strains of sel-12 mutants showed significantly elevated OCR, of approximately seven picomoles per minute, per worm.
Upon the addition of FCCP, there was an increase in the OCR of wild type, as well as sel-12 mutants, as expected. Wild type animals showed maximum OCR of approximately seven picomoles per minute, per worm. While sel-12 mutants had OCR of approximately 10 picomoles per minute, per worm.
Use live, well fed, and asynchronized animals in this assay, and avoid the transfer of eggs or carcasses into assay wells. Furthermore, the internal temperature of the instrument should not exceed 25 degrees Celsius. Bleach, FCCP, FUdR, and sodium azide are toxic.
Therefore, care should be taken to wear protective gloves during preparation of stock solutions and drug loading.
