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. Ins