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

Abstract

Source: Elettra Leo¹, Luiza H.D. Cardoso¹, Sabine Schmitt¹, Erich Gnaiger<sup>1

1</sup>Oroboros Instruments

This video explains how oxygen flux is used to assess mitochondrial respiration in living and permeabilized mammalian cells. It demonstrates the sequential use of digitonin, metabolic substrates, ADP, an uncoupler, and an inhibitor to characterize different respiratory states. The resulting oxygen flux profile reveals mitochondrial function and energy-coupling efficiency under defined conditions.

Protocol

1. Adding HEK 293T cells to the high-resolution respirometry chamber

1. Add mitochondrial respiration medium MiR05 into the clean Oroboros chamber. Close the chamber by inserting the stopper and siphon off the excess medium from the stopper receptacle using the suction system.
2. To add HEK 293T cells to the chamber by partial volume replacement, calculate the volume of the cell stock suspension required to reach an experimental cell concentration of 1 million/mL.
3. Remove the stopper from the chamber.
4. Remove a volume of MiR05 from the chamber corresponding to the calculated volume of the cell stock suspension using a pipette.
5. Resuspend ....

Results

Figure 1: High-resolution respirometry of HEK 293T cells. Trace of O₂ concentration [&#.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ADP	Merck, Germany	117105
Antimycin A	Sigma-Aldrich, USA	A8674	CAUTION H300 Fatal if swallowed, H400 short-term (acute) aquatic hazard
BSA fatty acid-free	Sigma-Aldrich, USA	A 6003	Bovine serum albumin
CCCP	Sigma-Aldrich, USA	C2759	Carbonyl cyanide m-chlorophenyl hydrazone CAUTION H301 + H311 + H331 Toxic if swallowed, in contact with skin or if inhaled
Digitonin	Sigma-Aldrich, USA	D5628	CAUTION H301 Toxic if swallowed
Malate	Sigma-Aldrich, USA	M1000	CAUTION H319 Causes serious eye irritation.
MiR05-Kit	Oroboros Instruments, Austria	60101-01	Mitochondrial respiration medium
Pyruvate	Sigma-Aldrich, USA	P2256	CAUTION H317 May cause an allergic skin reaction
HEK 293T cells	American Type Culture Collection	CRL-3216	Obtained through LGC Standards (Wesel, Germany)