High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

Summary

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes' (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

Abstract

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

Introduction

Mitochondria fulfill important roles in cellular energy metabolism, especially by using oxygen to produce adenosine triphosphate (ATP). They are implicated in cell death and in several human diseases. Mitochondrial oxidative phosphorylation (OXPHOS) combines electron transport along the electron transport chain with oxygen consumption and ATP synthesis. The mitochondrial tricarboxylic acid (TCA) cycle is involved in the conversion of proteins, carbohydrates and fats into energy rich compounds as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH₂). Electrons of the NADH and FADH₂ are then transferred to the respiratory....

Protocol

1. Cell Culture

1. Culture human hepatoma HepG2 cells⁶ in 25 cm² cell culture flasks in Dulbecco's Modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in an incubator (5% CO₂, 95% air) (seeding density: 1 x 10⁶ cells per 25 cm² cell culture flask, incubation time in the 37 °C incubator: 48 hr, cell density at confluency: 4-5 x 10⁶ cells per 25 cm² cell culture flask).
2. Perform the experiments when cells are 90% to 95% confluent.

2. High-....

Results

Determination of Optimum Digitonin Concentration for Cellular Permeabilization: Digitonin Titration Experiment

Digitonin titration is performed to determine the optimal concentration for permeabilization of HepG2 cells. For these experiments, digitonin is titrated in intact cells in the presence of rotenone, succinate (mitochondrial complex II substrate) and a saturating amount of ADP (to induce complex II-dependent state 3), and r.......

Discussion

The objective of the present protocol was to use high-resolution respirometry to measure mitochondrial respiratory chain complexes' (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity and mitochondrial outer membrane integrity.

There are some critical steps within the present protocol. First, cellular oxygen consumption rates are usually normalized to the number of cells (pmol/[sec x number of cells]). Therefore, before monitoring cellular oxygen consumption,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ADP	Sigma	A 4386	Chemical
Antimycin A	Sigma	A 8674	Chemical, dissolve in ethanol
Ascorbate	Merck	1.00127	Chemical
BSA	Sigma	A 6003	Chemical
FCCP	Sigma	C 2920	Chemical, dissolve in ethanol
Countess automated cell counter	Thermo Fisher Scientific	n/a	Automated cell counting instrument
Cytochrome c	Sigma	C 7752	Chemical
Digitonin	Sigma	D 5628	Chemical, dissolve in DMSO
DMEM	Gibco	31966021	Medium
EGTA	fluka	3779	Chemical
FBS	Gibco	26010-074	Medium component
Glutamate	Sigma	G 1626	Chemical
Hepes	Sigma	H 7523	Chemical
KCl	Merck	1.04936	Chemical
KH2PO4	Merck	1.04873	Chemical
K-lactobionate	Sigma	L 2398	Chemical
MgCl2	Sigma	M 9272	Chemical
O2k-Core: Oxygraph-2k	Oroboros Instruments	10000-02	High-resolution respirometry instrument
Oligomycin	Sigma	O 4876	Chemical, dissolve in ethanol
Penicillin-streptomycin	Gibco	15140122	Chemical
Sodium azide	Sigma	S2002	Chemical
Rotenone	Sigma	R 8875	Chemical, dissolve in ethanol
Succinate	Sigma	S 2378	Chemical
Taurine	Sigma	T 8691	Chemical
TMPD	Sigma	T 3134	Chemical
Trypsin	Sigma	T 4674	Chemical