The overall goal of the following experiment is to use microplate based oxygen consumption technology to assess the function of the electron transport chain, TCA cycle beta oxidation pathway, and substrate transporters. This is achieved by first preparing the substrate and injection solutions for the assay. The second step is to add the injection solutions into a previously hydrated tric assay cartridge to calibrate the oxygen consumption machine.
Next, prepare the mitochondrial substrate solutions and load these solutions in the cell culture plate. Spin the cell culture plate to allow mitochondrial adherence to the plate well before loading the plate into the Respira metric assay. Instrument results are obtained that show oxygen consumption rates based on mitochondrial respiration for each respiro metric assay.
The main advantage of this technique over existing methods like the Clark Electrode, is that microplate based technologies allow for more efficient and higher throughput acquisition of oxygen consumption with smaller amounts of mitochondria, generally individuals new to this method will struggle because of all the moving parts involved in preparing the solutions for the assay On the night before the experiment Hydrate tric assay cartridge in one milliliter of calibration solution in a non CO2 incubator at 37 degrees Celsius on the following day, take out all frozen stalks and thaw them in a 37 degrees Celsius bath or incubator. Prepare all substrate solutions and injection solutions and store them on ice. The next step is to program the multi-well oxygen consumption measurement machine to the proper mix weight and measure parameters.
Enter the analysis software and select the standard mode. Enter into the seahorse guest forum and click on the assay wizard. Once in the assay wizard, click on protocol to change the mix weight and measure parameters.
Label the background wells under the back correction tab and be sure to highlight. Do background correction. Label the conditions by first clicking on the groups and labels tab, followed by the group info tab.
After these parameters are entered, click end followed by save template. To begin this procedure, load the injection solutions into an assay run cartridge. Take care to inject the solutions into the proper ports.
Load the assay run cartridge into the machine with a notched corner to the bottom left. Start the calibration by first entering the analysis software followed by entering the standard mode. Enter into the seahorse guest forum.
Open the saved template under the XF Drive under the templates tab. Once open, click start to begin the calibration, which will take about 30 minutes. The mitochondria for this procedure were isolated from red skeletal muscle gently but thoroughly mixed the mitochondria substrate stock by stirring gently iterating the solution with a 200 microliter pipette tip that has had about three millimeters of its end cut off.
After determining the protein concentration of the mitochondrial stock, resus suspend 10 micrograms of mitochondrial protein per 200 microliters of the succinate rot known substrate mix. Mix the mitochondria substrate solution gently but thoroughly by stirring gently tating the solution with a 200 microliter pipette tip that has had about three millimeters of its end cut off in the same manner. Reeses Band, 14 micrograms of mitochondrial protein per 200 microliters of each of the remaining substrate mixes place all mitochondrial protein substrate mixes on ice.
Place a 24 well cell culture plate on ice and in triplicate load 50 microliters per well of each of the mitochondria substrate mixes. The developers of the microplate based tric assay recommend leaving a minimum of two blank wells with no mitochondria, preferably on either side of the cell culture plate. Spin the cell culture plate at 2000 G for 20 minutes at four degrees Celsius to adhere the mitochondria to the wells while the plate is spinning.
Warm up the substrate solutions in a 37 degree Celsius water bath. After the spin is complete, carefully load 450 microliters of each substrate solution on top of its respective wells. Be sure to load the plate at room temperature.
Load blank wells with 500 microliters of substrate blank. Wells designated for the electron flow assay should be loaded with the electron flow substrate while the coupled assay blank wells can be loaded with any coupling assay substrate. After 450 microliters of substrate is added to each, well view the layer of mitochondria in the well at 20 x magnification to ensure the mitochondria are dispersed evenly in a single monolayer.
As shown in this example, image wells that do not appear to have an evenly distributed monolayer can be removed post-oc. After confirming mitochondrial adherence, take the cell culture plate to the Respir metric assay instrument. Click okay.
The machine will eject the utility plate that was used earlier for calibration. Remove the utility plate and place the cell culture plate that contains the mitochondria on the tray. The blue notch on the cell culture plate should be placed in the bottom left corner of the tray.
Click continue to start the assay run, which takes about 40 minutes. After the run is complete, press eject on the screen to eject the cell culture plate. Once the plate is ejected, remove and discard the cell culture plate and cartridge.
Hit continue on the screen. The run will be automatically saved as an XLS file in the data file. Open this file and change the display from O2 to OCR by hitting the downward arrow under the Y one marker.
Next, change middle point to point to point rates under the rate data displayed as option. Click okay. To apply these changes, click the well group mode tab on the bottom left of the screen to group wells of similar conditions together.
Finally, click the sample mean standard error tab toward the middle of the screen to the left. These commands could also be set up before the assay. These tracings represent oxygen consumption rates or OCR versus time for four coupling assays.
The first panel represents state two, respiration or respiration in the absence of a DP.The second panel after injection of A DP represents maximal coupled respiration, also called state three respiration. The third panel after injection of oligo mycin. A represents respiration due to proton leak, also called state four oh respiration.
The fourth panel after injection of FCCP represents maximal uncoupled respiration, also called State three U respiration. The fifth panel after injection of antimycin A represents the inhibition of oxidative respiration. All mitochondrial states have minimal standard deviation due to thorough mixing of the mitochondrial stock and the mitochondria substrate mixes and the attainment of a single monolayer of mitochondria after the adherence spin.
In contrast, loading unequal mitochondria in each well and not attaining a single monolayer of mitochondria leads to increased standard deviation in each state, as indicated by the Blue trace, which shows OCR values above 1, 500 ole per minute, as well as decreasing respiration rates over the course of the measurement period exemplified by the drop in point to point rate values. Note that the Red trace shows consistent point to point rates and maximum OCR values are under 1, 500 ole per minute. Mitochondrial protein overload can also lead to the exhaustion of oxygen within the micro chamber of the well, thus preventing accurate measurement of OCR for each successive measurement as indicated by the Blue Trace, which shows O2 values approaching zero during the measurement period, as well as significantly reduced starting O2 values after each successive measurement.
While attempting this procedure, it's important to remember to have patients when mixing the mitochondria and the substrate solutions. Once these methods are mastered, additional experiments can be deformed in order to answer additional questions like, how does drug targeted treatments of a certain substrate transporter influence mitochondrial oxygen consumption?
