Interrogating real-time bioenergetic profiles of oxphos and glycolysis is emerging as a key factor in characterizing RPE health and function. High-resolution respirometry enables an efficient way of comparing the metabolic status of normal and diseased RPE. This technique has the advantage of simultaneous exploration of both oxphos and glycolysis through a measurement of the oxygen consumption rate, OCR and extracellular acidification rate, ECAR.
RPE cells are highly metabolically active cells and are one of the first cells to degenerate during AMD. Understanding their metabolic and mitochondrial function, makes it possible to develop novel drugs. To begin, add 100 microliters per well of the cell suspension in the human RPE medium to a final concentration of 20, 000 cells per 100 microliters in each well.
And ensure to leave the four corner wells empty. Pipette up and down multiple times to ensure a homogenous cell suspension, using a multi-channel pipette for ease and consistency. Add 100 microliters of media only to the four empty corner wells for background correction.
Leave the cell culture plate at room temperature for one hour to help minimize the edge effects. Then place it into the incubator with 5%carbon dioxide 37 degrees Celsius and humidified. Following overnight incubation, examine the cells under the microscope to check their morphology and pigmentation level before changing the media.
On subsequent examination days, ensure that the cells are confluent with a characteristic cobblestone-like morphology and acquire pigmentation over time. To ensure the hydration of the sensor cartridge the day before the assay, fill each well of the utility plate with 200 microliters of deionized water and place the sensor cartridge, submerged in water on the utility plate. Keep approximately 20 milliliters of the calibrant solution overnight in a 37 degrees Celsius humidified oven without carbon dioxide.
Turn on the high-resolution respirometry instrument and start the software to allow the instrument to stabilize at 37 degrees Celsius overnight. On the day of the assay, replace the water in the utility plate with an equal volume of warmed calibrant solution at least 45 minutes prior to running the assay. Warm 25 milliliters of the prepared Mito stress test assay media without phenol red to 37 degrees Celsius and vacuum filter the pH 7.4 adjusted media, using a tube top filter unit.
Remove the human RPE media from the cell culture plate and add 100 microliters of freshly prepared assay media. Then place the cell culture plate in a 37 degrees Celsius humidified oven without carbon dioxide for one hour before starting the assay. Each sensor cartridge has four reagent delivery ports per well for injecting the test compounds into cell culture plate wells during the assay.
Prepare three milliliters of the Tenex drug solutions each, by diluting the drug stocks in their respective assay media. Next, pipette 20 microliters of the Tenex drug stock into Port A, 22 microliters into Port B and 25 microliters into Port C to achieve the specified final drug concentration in each well. Next in the analysis software, open the templates tab, select Mito stress test and fill out the group definitions.
Input details on the injection strategy for the Mito stress test drugs. Then input details on the different experimental groups in the assay for control or treatment. Input details on the assay media for adding different supplements and their specific concentrations to the base assay media.
And finally, add the cell type. Click on the next tab and then on Plate Map to assign different groups under examination to their specific location on the 96 well plate. On completing the Plate Map, click on the protocol tab to review the instrument protocol for the default Mito stress test protocol.
Then click on run the assay, and insert the sensor cartridge, submerged in the calibrant solution in the utility plate for calibration. This process takes around 25 minutes and each biosensor gets calibrated independently, based on the sensor output measured in the calibrant solution of known pH and oxygen concentration. On the completion of calibration, remove the utility plate and insert the cell culture plate.
After the baseline measurement, the instrument automatically injects the Port A drug solution into each well which follows three loops of mixing and measuring, three minutes each. The same pattern occurs after each subsequent drug injection in the other ports. Once the run is complete, remove the cell culture plate and sensor cartridge.
For quality control purposes, ensure that all the drug ports and the sensor cartridge have been injected by examining the ports for no residual drug remains. Next, examine the cells in the cell culture microplate under the microscope to ensure the confluent monolayer of cells. Then discard the assay media and replace it with 60 microliters of one x lysis buffer in each well.
Wrap the edges of the plate in parafilm to prevent evaporation, and place it in a minus 80 degrees Celsius freezer to aid in cell lysis overnight before quantification of the protein content, using the BCA assay. Per data analysis, normalize all data by dividing the oxygen consumption rate or OCR and extracellular acidification rate or ECAR values by the micrograms of protein in each well. Then export the Mito stress test report generator, which utilizes Excel macros to automatically calculate the Mito stress test parameters using the data analysis software.
By following the same steps as demonstrated for the Mito stress test, the glycolytic stress test can be performed, except for the assay media supplements and drug injections which are different as shown in table one and table two. The instrument simultaneously measures both OCR and ECAR for each run. For the Mito stress test, perimeter calculations are based on OCAR readings, whereas for the glycolytic stress test, parameter calculations are based on ECAR readings.
Here is an OCR curve generated from performing the Mito stress test on primary human RPE cells. Calculations of Mito stress test parameters are displayed as bar graphs. Similarly, this is the ECAR curve, generated from performing the glycolytic stress test on primary human RPE cells and the calculations are shown as bar graphs.
To optimize the port B drug injection for the Mito stress test, the efficacy of two uncoupling agents in increasing OCR in primary human RPE cells was compared. BAM15 was found to be superior to FCCP in enhancing mitochondrial respiratory capacity as seen by the significantly higher maximal respiration and spare respiratory capacity with BAM15, compared to FCCP. It is important to remember to hydrate the sensor cartridge the day before running the assay.
This technique allows researchers to characterize the bioenergetic profiles of RPE cells better and understand how RPE cells exhibit metabolic flexibility in response to different pathogenic stimuli.
