In this work, we describe a modified protocol to test mitochondrial respiratory substrate flux using recombinant perfringolysin O in combination with microplate-based respirometry. With this protocol, we show how metformin affects mitochondrial respiration of two different tumor cell lines.
Because it tests mitochondria substrate flux that can be affected with different pathologies or treatments. For example, today we will use it to reveal cancer cell response to Metformin treatment. It requires a minimal number of cells while having sufficient replicates and appropriate control for each distinct material.
The analyzer is for research use only. This technique can identify errors in mitochondrial metabolism and can be used to evaluate warriors drugs affecting mitochondria. Demonstrating the procedure will be Karolina Vaneckova an undergraduate student and research technician from my laboratory.
To begin seed A549 cells at a density of 20, 000 cells per well in columns, two to 11 of a seahorse XF 96 cell culture micro-plate. Leaving columns one and 12 empty as background wells. Fill the blank Wells with an equal volume of the cell culture medium, then incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for cell attachment.
After three to four hours, add 100 microliters of cell culture medium in the Wells. Treat the experimental group columns seven to 11 with one millimolar Metformin and the control group with an equal volume of sterile distilled water, then return the plate to the incubator. For hydrating the sensors, pipette 200 microliters of sterile water per well into the utility plate.
Then carefully return the sensor cartridge while immersing the sensor in water. Incubate the cartridge in a carbon dioxide free incubator at 37 degrees Celsius till the next day. Switch on the analyzer and the control unit.
Start the instrument control and data acquisition software, and design the assay protocol as described in the text manuscript. Under group definitions, create four injection strategies where port A differs according to the injected substrate and name the strategies after the substrates or abbreviations. Assign Oligomycin to port B, FCCP to port C and Rotenone Antimycin A mixture to port D.Create and name eight groups, under the plate map, assign groups to the corresponding Wells.
Then save the protocol as a ready to use template. Leave the analyzer switch on to allow the temperature to stabilize overnight. The next day, discard the water from the utility plate and add 200 microliters of the pre-warmed calibrant per well in the utility plate.
Return the cartridge to the carbon dioxide free incubator until the time of the assay. Maintain a source of humidity inside the incubator and turn off or reduce the fan speed to a minimum, to avoid rapid evaporation of the calibrant. Prepare five milliliters of the working solution of the substrates and inhibitors with pre-warmed two times mitochondrial assay solution, 5%BSA and sterile water as described in the text manuscript.
Next, load the injector ports with substrate and inhibitors as described in the tax manuscript. For calibration under the run assay tab, click on the start run to start the assay. Insert the loaded censor cartridge and wait for the calibration to complete.
Prepare a 20 milliliters of assay medium, by mixing two times mitochondrial assay solution, sterile water and 5%BSA in a 50 milliliter tube. Add two microliters of 10 micromolar recombinant perfringolysin O, to attain a concentration of one nanomolar. And re-suspend the mixture with gentle pipetting, avoiding shaking and vortexing.
Incubate the tube at 37 degrees Celsius until use. Use a multi-channel pipette to wash the cells and the empty blank Wells two times, using pre-warmed calcium and magnesium free PBS solution. Discard the PBS and add 180 microliters of the pre-warmed assay medium for cell permeabilization.
Immediately after permeabilization replace the utility plate of the calibrated sensor cartridge with the cell plate containing permeabilized cells and start the measurement. The treatment group had a higher rate of succinate induced respiration. The response of A549 cells to Metformin treatment was higher than Hep G2.For the pyruvate malate induced respiration, A549 cells showed increased induction compared to Hep G2 cells between five to 15 minutes.
Similar results were obtained for glutamate malate induced respiration and palmitoylcarnitine malate induced respiration. To attain the right concentration of substrate and inhibitors. The right volume must be loaded into the right port.
When a change in a metabolic pathway of a certain substrate is identified, it will be necessary to assess the function of the enzymes and transporters involved in that pathway.
