Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Summary

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Abstract

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Introduction

Mitochondria are organelles, approximately the size of bacteria, found in eukaryotic cells. They are unique organelles because they contain DNA and have two membranes, an outer and inner one. Mitochondria's outer and inner membranes are separated by an intermembrane space, and the inner membrane folds into structures called cristae around the innermost compartment, called the matrix. These cristae increase the inner membrane's surface area so that multiple processes that use the cristae can occur simultaneously. While mitochondria are involved in many cellular functions such as controlling apoptosis and housing multiple metabolic pathways, their vital role in ....

Protocol

Lizards were euthanized by CO₂ asphyxiation followed by immediate decapitation in accordance with policies outlined by The Office of Animal Laboratory Welfare and Elon's Institutional Animal Care and Use Committee guidelines.

1. Isolation of mitochondria⁶

NOTE: Keep all solutions cold (Table 1) and samples on ice throughout these steps.

1. Remove the liver, weigh it, and then rinse it .......

Discussion

Measuring mitochondrial function is useful when comparing different samples, such as disease versus non-disease states, different tissue types from the same animal, or between different sample types. We used the later comparison to test our hypothesis that there is a metabolic consequence to hybrid tree lizards that have introgressed mitochondria. There are a variety of ways to ascertain mitochondria function experimentally, including quantification of Δψ, total ATP content, ATP production, and respiratory cont.......

Materials

Name	Company	Catalog Number	Comments
96-well black/optical bottom plates	Thermo Fisher	265301	Untreated black-wall plates with clear bottoms.
ADP	Sigma	A2754	Dilute 100 µM stock with EB immediately before use.
BSA	Thermo Fisher	BP1600-100	Make 2 mg/mL stock in water for protein assay.
Dulbeccos 1x PBS (-/-)	Sigma	D8537	Make sure the PBS is without Mg2+ or Ca2+ ions.
EGTA	Sigma	E3889
K2HPO4	Sigma	P3786
KH2PO4	Sigma	P0662
L-glutamic acid	Sigma	G1251
L-glutamic acid potassium salt	Sigma	S372226
L-malic acid	Sigma	M8304
L-malic acid mono-potassium salt	Sigma	49601
MitoXpress oxygen consumption kit	Agilent	MX-200-4	Kit contains probe stock and HS mineral oil.
MOPS	Sigma	M3183
Protien Assay Dye (5x)	BioRad	500-0006	Any protein assay can substitute.
R version 3.3	R Core Development Team 2016
Thermomax microplate reader EnSpire Multi-mode Plate reader and software	PerkinElmer		Standard fluorescent plate-reader
Trisma base	Sigma	T6066	Any version of Tris base can be utilized.