The overall goals of this procedure are to analyze the metabolic activity of a biological sample and to determine the extent that anaerobic pathways contribute to the cellular energy production within the sample. This method can help answer key questions in the pharmaceutical field about the extent of mitochondrial toxicity during drug development. The main advantage of this technique is that it facilitates the simultaneous measurement of oxygen consumption and heat production in a cell sample of interest.
So this method can provide insight into treatment planning during pharmaceutical development. It can also be applied to other fields, such as cyrobiology in which the effects of metabolic preconditioning on cryopreservation can be assessed. To prepare the respirometer, first add two-point-two milliliters of DMEM to each respirometer chamber and fully insert the stoppers.
Adjust the stoppers with a stopper-spacer tool to allow optimal oxygen diffusion to the medium, and set the chambers to continuously stir the samples at 750 rpm at 37 degrees Celsius until the oxygen concentration and the oxygen flux are stable. Confirm that the respirometer software is programmed to account for the oxygen solubility of the medium used in the experiment. Then select a stable portion of the oxygen concentration trace and calibrate this portion for 100%air saturation.
Now, close the stoppers, taking care that no bubbles are present in the chambers. After about 10 minutes, a stable oxygen flux should be observed. To prepare for calorimetry, first add two-point-five milliliters of de-ionized water to the reference ampule, then tighten the cap.
Using an air pump, remove external debris from the ampule, then slowly lower the ampule to position one in the reference channel of the calorimeter. The ampule will remain at position one until the ampule with the cell sample has been prepared. DMEM, used for calorimetry, should be equilibrated in a 37-degree Celsius incubator with a humidified atmosphere and 5%CO2.
Begin the experiment by adding three milliliters of DMEM to a 100-milliliter plate for each calorimetric ampule that will be used, and place the plate in the incubator until the sample is ready for calorimetry. While the medium is equilibrating, use an inverted microscope to select a plate with HepG2 cells that are between 60 and 80%confluent and are homogeneously distributed. First, remove the cell culture medium and wash the cells two times with 10 milliliters of PBS.
Add three milliliters of pre-warmed trypsin at 37 degrees Celsius and incubate the plate at 37 degrees Celsius. After seven to 10 minutes of incubation, neutralize the enzymatic reaction with the three milliliters of 37-degrees Celsius DMEM and use a five-milliliter pipette to gently dissociate the cells into a single-cell suspension. Transfer the cells into a sterile 15-milliliter conical tube for centrifugation, and re-suspend the palette in approximately five milliliters of fresh DMEM.
Pipette the cells thoroughly to disaggregate any cell clusters, and after counting, concentrate the cells to approximately two millions cells per 50 microliters of medium. The reproducibility of this method is dependant upon on consistent and accurate cell counts. It is critical to ensure the sample is homogeneous and that cells are not clumped at any point during the analysis.
Then, store the cells on ice until ready to perform respirometric and calorimetric measurements. Before the calorimetric measurement, confirm that the calorimeter is connected to the computer and that the calorimetric signal is stable and in micro-watts. Dilute the cells to 100, 000 cells per milliliter in the equilibrated medium that was placed in the incubator prior to sample preparation.
Then add two-point-five milliliters of cell solution to each ampule, confirming that a sufficient volume of air is maintained between the lids of the ampules and the medium to allow for gas diffusion. Immediately tighten the caps and clean the sealed ampules with air to remove any external debris. Then slowly lower the sealed ampule with the cell sample to position one of the calorimeter.
Thoroughly mix the sample to ensure a homogeneous solution, and inject two million cells into each closed chamber of the respirometer under continuous stirring. After 15 minutes at position one in the calorimeter, slowly lower both the reference ampule and the ampule with the cells to position two and record the time of lowering. 10 to 15 minutes after the cells have been injected into the respirometer, a stabilization of the oxygen flux and a steady decrease in the oxygen concentration should be observed.
To determine leak respiration, inject one microliter of four milligrams per milliliter oligomycin into each chamber and allow the oxygen flux to stabilize for about 10 minutes, recording the stable leak respiration rate as just demonstrated. Then, to determine the maximal flux, inject two microliters of zero-point-two millimolar FCCP into the chamber, allowing the oxygen flux to stabilize after each injection, and continuing the titrations until the maximal flux is reached. Record the stable respiration rate during the maximal flux.
When the respirometer chamber is operating prior to sample measurement, both the oxygen concentration and oxygen flux traces should be stable. After the ampule has been lowered to position two, a time period can be identified in which the heat output is stable, and a corresponding time can be identified in which the oxygen flux is stable, to allow calculation of the calorespirometric ratio. If the HepG2 cells are cultured in the absence of glucose and galactose is supplemented to increase the mitochondrial involvement, the cells do not proliferate inside the calorimeter, but instead decrease their metabolic activity after about 30 minutes in the ampule, restricting the calorespirometric calculation to the earliest time point at which the heat dissipation was measured.
It is critical to record the time the cells are pipetted into the ampule so that the measurements of the heat production and the oxygen consumption are collected at identical time points, as it takes approximately 15 minutes for the oxygen flux to stabilize and the oxygen concentration to steadily decline. As the stabilized oxygen flux of the cells serves as a surrogate for the oxygen consumption when calculating the calorespirometric ratio, additional titrations can be performed to assess the leak respiration, which is indicated by a drop in the oxygen flux and the maximal uncoupled respiration, which is indicated by a failure to increase the respiration with successive FCCP titrations. Once mastered, this technique can be completed in less than four hours if it is performed properly.
While attempting this procedure, it is important to remember that other cell lines may have different metabolic properties. Therefore, each new cell line must be optimized for both it's calorimetric and respirometric measurements. After watching this video, you should have a good understanding of how to prepare HepG2 cells for calorespirometry to ensure correct instrumental calibration, and to properly conduct a calorespirometric experiment.
