This protocol is significant for determining the glycolytic function and mitochondrial respiration in Candida albicans. The main advantage of this technique is that it allows for mitochondrial function to be measured without the need to purify mitochondria from Candida albicans. This method can be optimized to investigate the effects of genetic manipulation or chemical modulators on mitochondrial and glycolytic pathways in Candida albicans or other fungal pathogens.
This technique is relatively simple. Or depending on the organism, their use may help to optimize the cell number and the concentration of mitochondrial inhibitors in their assay. In a laminar flow hood, dissolve Poly-D-Lysine in tissue grid water to a final concentration of 50 micrograms per milliliter.
Mix the solution well and aliquot it into 1.5 milliliter microcentrifuge tubes making sure to aliquot at least 1.3 milliliters per tube. Add 50 microliters of this mixture to each well of the assay plate, cover the lid and incubate at room temperature for one to two hours. Then aspirate the solution.
Rinse the wells of the assay plate once with 500 microliters of sterile tissue culture grade water, open the lid and allow the wells to air dry. If not using the plate the day it is prepared, store it at four degrees Celsius for a maximum of two to three days. The day before the experiment, open the extra flux assay kit and remove the contents.
Place the sensor cartridge upside down next to the utility plate. Fill each well of the utility plate with one milliliter of calibrant and then place the sensor cartridge back. Make sure the sensors that contain fluorophores are submerged in the calibrant.
Incubate the sensor cartridge at 37 degrees Celsius in a non-carbon dioxide incubator overnight. The day before the assay, inoculate the prepared C albicans in the YPD broth and grow overnight at 30 degrees Celsius in a shaker at 200 rpm. On the day of the assay, dilute an appropriate number of cells in the assay medium to yield a final concentration of 100, 000 cells per 100 microliters.
Add 100 microliters of the diluted cells into each well of the assay plate except wells A1, B4, C3, and D6.To these wells, add only 100 microliters of assay medium for background correction. Incubate the plate at 37 degrees Celsius in a non-carbon dioxide incubator for 60 minutes to let the cells adhere to the plate surface. Prepare the compounds for the mitochondrial function assay at 10X concentration in the corresponding assay medium as outlined in the text protocol.
Add 50 microliters of SHAM into port A, 50 microliters of Oligomycin into port B, 62 microliters of potassium cyanide into port C, and 68 microliters of Antimycin A into port D.Next, prepare the compounds for the glycolytic stress assay at 10X concentration in the corresponding assay medium as outlined in the text protocol. Add 50 microliters of glucose to port A, 55 microliters of Oligomycin into port B, 62 microliters of 2-DG into port C, and 68 microliters of Antimycin A into port D.Before the assay, open the extra flux analyzer and set up the assay template by using the assay wizard tab. Follow the step-by-step instructions and fill out all of the information that pop out during the setup.
Generate a group layout and set up the protocol as outlined in the text protocol. Save these layouts before the assay. At the time of the assay, restore the saved protocol by opening the corresponding file in the open file option in the assay wizard tab.
Then load the 10X compounds into the respective ports of the hydrated sensor cartridge containing the calibrant. Load the sensor cartridge into the carrier tray of the extra flux analyzer. Start the calibration by clicking the start button on the screen.
Next, gently add 350 microliters of the assay medium to the cell plate along the side of the walls to minimize cell disturbance bringing the final volume to 450 microliters. Replace the utility plate containing the calibrant with the assay plate and continue the assay. Once the assay is completed, remove the sensor cartridge and the plate.
Save the file in the appropriate destination folder. In this study, the bio-energetic functions of the C albicans are assessed by extra flux analyzer. A mutant lacking the mitochondrial protein mam33 is also included along with its complement strain to study the effects of the deletion of a mitochondrial protein on oxygen consumption rate and extracellular acidification rates.
The C albicans wild type shows an oxygen consumption rate of 145 picomoles per minute which is well within the optimal range for any extracellular flux assay. The basal oxygen consumption rate of the wild type, mam33 mutant and mam33 complemented strains show no differences. Similarly, no significant differences are seen in the basal extracellular acidification rates between the strains.
However, by inhibiting complex-IV with potassium cyanide, the wild type and mam33 mutant, mam33 complemented strains show a significant shift toward the glycolysis. The mam33 mutant strain however failed to show a compensatory glycolytic shift suggesting that the compound-IV dependent pathway is impaired in this strain. In the glycolytic stress test, cells are starved of glucose for one hour and the basal oxygen consumption rate and extracellular acidification rate is measured.
Upon starvation, the mam33 mutant strain show significantly lower oxygen consumption and extracellular acidification rates compared to the other strains. This suggests an impaired utilization of glucose for both respiration and glycolysis when cells are forced to the starvation condition. After injecting glucose, all strains show stimulation of both their oxygen consumption rate and extracellular acidification rate.
The coating of assay plate allows Candida albicans cells to adhere to the plate. Improper adherence of Candida albicans cells will affect the assay result. After the development of this technique, it has been employed in a wide range of cell types and diseases addressing how mitochondria function during physiological and pathological conditions.
For example, employing patient-derived and control cells in this assay, it is possible to address the specific questions of how mitochondrial function is altered. Care should be taken while preparing mitochondrial inhibitors such as potassium cyanide and antimycin A because these compounds are extremely poisonous.
