Our research uses drosophila melanogaster as experimental model to understand mitochondrial function in disease and chemical toxicity. Some questions we want to answer are, how does PQ one patient affect mitochondrial function and how is this linked to Parkinson's disease? How can we prevent or evert the mitochondrial dysfunction associated with this mutation?
High resolution respirometry requires the desired training for precise operation in data analysis. In addition, the system user doesn't allow working with several samples simultaneously and is not educated for high throughput analysis. Since it's only possible to run two samples at the same time, the experimental design must be well delineated.
Our findings in case progressive environment of the mitochondrial function we facing in pink null mutants which is characterized by decrease in oxphos C1 linked and ETS C1 and C2 linked and a metabolic shift in ATP production from ox oxidative to pathways. These findings are closely related to Parkinson's mutation disease pathophysiology. One of the key points of high resolution he respiratory is it's ability to provide, direct and accurate measurements of oxygen consumption along for mitochondrial function and cellular metabolism.
Also, it's versatile. It can be used for wide range of several types, including isolated mitochondria. Considering that mitochondrial dysfunction is a common feature of neurodegenerative diseases such as Parkinson's disease.
In the future, we will focus on investigating how mitochondrial function and energy elevation could be important targets for therapies directed to neurodegenerative diseases. To begin, add two milliliters of MIRO five buffer to the Arobaros chamber. Cover the chamber with a stopper without introducing air bubbles.
Then carefully pull the stopper to form a single air bubble. Open the Arobaros DAT Lab software In the Block Temperature field enter a temperature of 25 degrees Celsius and click on Connect to XI GRA 2K. Click and select the folder where the calibration is to be saved and click on Save.
When a new window opens name the experiment and the samples. Then click on Save. Next, go to the layout menu and choose the first option, one, calibration experiment.
Graph three, temp. When the red line shows a standard straight line with points around zero picomol select two points, one for chamber A and the other for chamber B, select oxygen concentration on the right of the screen. Select the marked point and copy the temperature and barometric pressure values relative to the point.
Paste these values in the boxes below and click on calibrate and copy to clipboard. Then go to the file menu and select save and disconnect. After calibrating polaro graphic oxygen sensors, remove the stoppers and open the chambers.
Wash the stoppers and chambers with 100%ethanol, 70%ethanol and distilled water. Transfer the flies into a micro centrifuge tube. Add 200 microliters of chilled MiRO5 buffer into the tube and homogenize the flies by applying gentle pressure with four to six strokes of the pestle.
Using a micro pipette, transfer the homogenized fly suspension into the chamber. Cover the chamber with a stopper without introducing air bubbles. Next, click on the layout menu and select option five Flow by corrected volume"Another new window will open here in the space Experimental Code"name the experiment in the sample field.
Name each sample in its respective chamber A and B.Then set the unit field to unit and in the concentration field, define the chamber volume as one per milliliter. To start the high resolution respirometry ensure that the oxygen flux signal is steady on the positive value. Add digitonin to permiabilize the mitochondrial membrane.
Then add pyruvate, malate and proline substrates until the oxygen flux increases and stabilizes. Press the F4 key on the keyboard and enter the name of the reagent to mark the event. Add a ADP to couple the mitochondrial respiratory chain and wait for the increase and stabilization of the oxygen flux.
Then add succinate and measure the oxygen flux to inhibit A TP synthase add Oligomycin and examine for a decrease in oxygen flux. After the red line stabilizes uncouple the mitochondrial electron transfer using 0.25 micromolar FCCP until maximum oxygen consumption is reached, demonstrated by the rise of the red line. Next, add rotenone complex one inhibitor and wait for the decrease and stabilization of the oxygen flux.
Then add melanate complex two inhibitor and measure the oxygen flux. Finally, add antimycin complex three inhibitor and wait for the decrease followed by the increase and stabilization of the oxygen flux. Click on the Graph Pad Prism Software to extract the oxygen flux values from the graphs.
Oxphos C1 refers to the value of oxygen flux after adding ADP. Then use the given formula to calculate oxphos C2.Using the following equation, calculate the ETS C1 and C2 oxygen values for the uncouples and inhibitors. Then calculate the ATP synthesis as the difference between the oxphos and leak.
Finally, calculate the oxphos by leak ratio to determine the respiratory control ratio. The oxygen flux in both oxphos C1 and oxphos C1 and C2 states was significantly reduced in pink 1B9-null flies compared to control flies. Additionally, the oxygen flux in ETS C1 ETS C2 and ETS C1 and C2 state were lower in pink 1B9 null flies indicating an impaired electron transfer system.
Compared to the control flies. The disruption in the flux of electrons affects the oxphos process leading to reduced A TP synthesis in the pink 1B9-null flies. The reduction in the respiratory control ratio of null flies suggests mitochondrial uncoupling, indicating that the mitochondria were less efficient at utilizing oxygen and producing ATP.
