We developed a new method using a seahorse XF analyzer to directly measure the oxygen consumption rate. But common proxy for mitochondrial function in acute slide slice from adult mice. This method measures a cellular by energetics in punctures from anatomically defined brain structures.
Using acute slices more closely mimics the physiological cellular environment which cannot be achieved the ways isolated mitochondrial or culture cells. This method will be of broad interest to researchers working in the field of Parkinson's disease and Huntington's disease. To begin, open the extracellular flux assay kit and remove both the sensor cartridge and the utility plate.
Then place the sensor cartridge aside and do not touch the sensors. Next, add 600 microliters of calibrate solution to each wall of the utility plate. Place the sensor cartridge on top of the utility plate and submerge the sensors in the calibrate solution, ensuring that the triangular notch of the utility and sensor cartridge plate are correctly aligned.
Afterward, seal the extracellular flux assay kit with sealing film to prevent evaporation of the calibrate solution and then place it in a 37 degree Celsius incubator not supplemented with carbon dioxide or oxygen, overnight. Open the eyelet capture micro plate and take out the eyelet plate for tissue sitting. Then warm an appropriate volume of pre-oxygenated modified artificial cerebral spinal fluid buffer to 37 degree Celsius in a 50 milliliter tube.
Next add BSA to a final concentration of four milligrams per milliliter to prepare the respiration buffer. Add 625 microliters of respiration buffer to each well of the eyelet plate carefully while avoiding shaking the plate, ensuring no air bubbles are present in the buffer of each well. Immediately dissect the brain in 10 milliliters of ice-cold pre-oxygenated cutting solution.
Then, using a vibratome section coronal striatal slices, following the manufacturer's instruction at a thickness of 150 micrometer in ice-cold pre-oxygenated cutting solution. Next, recover the slices in 50 milliliters of oxygenated artificial cerebral spinal fluid. And keep them in solution for up to 30 minutes at room temperature.
After recovery, transfer the slices to a 35 by 10 millimeters Petri dish with five millimeters of respiration buffer. Using a stainless steel biopsy punch create a circular piece of tissue in the desired area of the sliced brain by gently pressing down the punch while keeping the slice in the buffer. Then remove the rest of the tissue, lift the punch away and remove the circular piece of tissue into the buffer.
Next, cut the very end of a one milliliter pipette tip to make a hole with a one-point-five to two-point-zero millimeter diameter and use it to hold and transfer the punched slice to the top of the capture screen. Suction one piece of punched brain tissue and carefully place the tissue onto the mesh side of the capture screen, a circular piece of plastic with the mesh attached to one side. Using a paper tissue gently dry the capture screen and remove the moisture, which allows the tissue to become sticky and attached to the center of the mesh.
Next, hold the capture screen slice-side down with tweezers and place it into one of the wells of the incubating eyelet plate. Then incubate the eyelet plate at 37 degrees Celsius in an incubator for at least 30 minutes to allow temperature and pH equilibration before running the assay. Dilute the desired compounds and modified artificial cerebral spinal fluid without BSA to the final stock concentration of the working concentration for ports A to D, respectively.
Gently preload 75 microliters of the diluted compounds into the appropriate injection ports of the sensor cartridge by placing the tips halfway into the injection ports at a 45 degree angle with the tip against the wall of the injection port, as complete insertion may cause compound leakage through the port. Afterward, withdraw the tips from the ports carefully without creating air bubbles and do not tap any portion of the cartridge to avoid alleviating air bubbles. Then visually inspect the injection ports for even loading ensuring all liquid is in the port and no residual drops are present on top of the cartridge.
After placing the sensor cartridge onto the utility plate put it into an incubator for 30 minutes to allow it to heat up to 37 degrees Celsius while handling carefully by only holding onto the utility plate and moving as little as possible. First load the assay template in the software and then press the green start button. Then load the sensor cartridge on the utility plate into the instrument tray, ensuring that the plate sits correctly and is flat, loading the drug-filled sensor cartridge into the analyzer for calibration.
Afterward, follow the instructions on the screen in order to calibrate. And equilibrate the sensors. Once the equilibration step is done, remove the calibration plate and replace it with the eyelet plate containing the mesh and tissue slices.
Then measure the oxygen consumption rate in each well of the plate, using the assay protocols as described in the manuscript. Following, analyze the oxygen consumption rate measurement data and the coupling efficiency data. Oxygen consumption rates for different thicknesses and punch sizes of slices in the control group showed stable basal respiration over the whole measurement and were proportional to the volume of the slice.
Furthermore, oxygen consumption rates were relatively stable for five hours with less than 10%rundown. The mitochondrial coupling efficiency was compared and the slice at the 150 micrometer thickness and a 1.5 millimeter punch size showed the highest coupling efficiency. The oxygen consumption rates were measured for young and old groups of Pink1 knockout and their age-matched wild-type mice.
The basal oxygen consumption rate was similar in both the knockout and wild-type groups for young mice. However it decreased for the knockout mice in the old group. However, the mitochondrial dysfunction in the knockout mice was observed for the young age group, indicated by the decreased coupling efficiency caused by the knockout of Pink1.
The critical steps in this protocol includes preparing brain slices, transferring them to the top of capture screen, placing slides into wells and the keeping them attached to the capture screen during the measurements.
