Oxygen Flux Measurements to Evaluate Mitochondrial Respiration in Living and Permeabilized Cells

Load a mammalian cell suspension into the respirometer chambers containing media, and close them with stoppers to create a closed system for oxygen measurement.

A sensor within each chamber measures the oxygen concentration, from which the oxygen flux is calculated.

Inject digitonin through the stopper capillary to permeabilize the plasma membranes, allowing cytosolic components to diffuse out.

This reduces endogenous substrate availability for the mitochondrial electron transfer system, lowering the oxygen flux.

Introduce pyruvate and malate, which enter the mitochondria and generate NADH.

This NADH donates electrons to Complex I of the ETS. This initiates electron transfer and increases the oxygen flux.

Inject ADP to initiate ATP synthesis, which further elevates the oxygen flux.

Next, titrate an uncoupler stepwise to decrease the pH difference between the matrix and the intermembrane space, maximizing the oxygen flux.

Finally, inject antimycin A to inhibit Complex III, halting electron transfer and minimizing the oxygen flux.

This flux profile reveals mitochondrial respiration in permeabilized cells.

To begin, add mitochondrial respiration medium MiR05 into the clean Oroboros chamber. Insert the stopper to close the chamber and siphon off the excess medium from the stopper receptacle using the suction system.

Before adding the HEK 293T cells, calculate the volume of cell stock suspension needed to achieve an experimental concentration of 1 million cells per milliliter.

Then, remove the stopper from the chamber and place it on the rack. Using a pipette, remove a volume of MiR05 Vout from the chamber corresponding to the calculated volume of the cell stock suspension.

After thoroughly resuspending the cell stock suspension using a pipette, add the calculated volume to the chamber.

To close the chamber, insert the stopper, ensuring that no bubbles are trapped inside. Siphon off any excess liquid from the stopper receptacle with the suction system.

Wait for the stabilization of oxygen flux, which typically takes about 15 minutes.

The traces of oxygen flux are displayed in the upper panel for both chambers, while the oxygen concentration is displayed in the lower panel.

To prepare the microsyringes, select and clean the appropriate one based on the type of chemical and volume to be titrated. Place the microsyringe on the syringe rack.

Now, load the syringe with the chemical according to the respiratory substrate-uncoupler-inhibitor titration or SUIT protocol, avoiding gas bubbles. Fill the syringe with at least 1 microliter more than the required volume.

Press the plunger until the desired mark on the glass barrel is reached, ensuring that a small drop appears at the needle tip. Wipe off the drop on the wall of the chemical vial before proceeding with titration.

When performing the titration, the needle should be inserted completely through the stopper capillary until the termination of the glass barrel is in contact with the stopper, and the chemical must be injected swiftly into the chamber.

To start this particular SUIT protocol, add digitonin to an experimental concentration of 5 micrograms per milliliter in both chambers. Then, click on OK in the event window to set the corresponding event for both chambers.

Siphon off excess liquid from the stopper receptacles. Wait for the stabilization of oxygen flux and record for at least 2 minutes.

Titrate pyruvate into both chambers, followed by malate to reach the respective experimental concentrations. Click OK in the event window.

Now, siphon off excess liquid from the stopper receptacle, wait for flux stabilization, and record for about 2 minutes.

Then, add ADP to an experimental concentration of 2.5 millimolar and click OK in the event window.

After clearing the excess liquid, wait for flux stabilization. The flux may take up to 20 minutes to stabilize.

Then, repeat the process with CCCP in 0.5 micromolar increments to reach maximum flux.

Add antimycin A to reach an experimental concentration of 2.5 micromolar.

After reaching stabilization of the flux, click on the button labelled Stop measurement.

For data analysis, switch the layout from overlay to separate chambers. Set marks on stable and representative sections of the flux plot, avoiding titration artifacts.

Select Marks from the top menu, then choose Mark Statistics. Select the median from the drop-down menu “statistics mode” and click on Copy to Clipboard to transfer the data for further analysis.