Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Summary

In this work, we describe a modified protocol to test mitochondrial respiratory substrate flux using recombinant perfringolysin O in combination with microplate-based respirometry. With this protocol, we show how metformin affects mitochondrial respiration of two different tumor cell lines.

Abstract

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.

Introduction

Microplate-based respirometry has revolutionized mitochondrial research by enabling the study of cellular respiration of a small sample size¹. Cellular respiration is generally considered as an indicator of mitochondrial function or 'dysfunction', despite the fact that the mitochondrial range of functions extends beyond energy production². In aerobic conditions, mitochondria extract the energy stored in different substrates by breaking down and converting these substrates into metabolic intermediates that can fuel the citric acid cycle³ (Figure 1). The continu....

Protocol

1. One day before the assay

1. Preparation of reagents and substrates.
   1. Mitochondrial assay solution (MAS): Prepare stock solutions of all reagents as described in Table 1. Warm the stocks of mannitol and sucrose to 37 °C to dissolve completely. Mix the reagents to prepare 2x MAS, then warm the mixture to 37 °C. Adjust the pH with 5N KOH to 7.4 (~7 mL), then add water to bring the volume up to 1 L. Filter-sterilize and store the aliquots at -20 °C until the measurement day.
   2. Bovine serum albumin (5% BSA): Dissolve 5 g of BSA in 90 mL of prewarmed sterile water on a magnetic stirrer and avoid shaking. A....

Results

Start by normalizing the results to the second measurement of baseline respiration to show values as oxygen consumption rate percentage (OCR%). The results of the assay are shown in Figures 5, Figure 6, Figure 7, and Figure 8. It is important to assign the proper background wells for each group and inactivate the background wells of other groups. Fi.......

Discussion

This protocol is a modification of previously published studies^7,8,9,10 and the product user guide. In contrast to the manufacturer's protocol, 2x MAS is used instead of 3x MAS, since 2× MAS is easier to dissolve and does not form precipitations after freezing. Frozen 2x MAS aliquots can be stored up to six months and show consistent results. Another difference is including ADP in the .......

Materials

Name	Company	Catalog Number	Comments
Adinosine 5′ -diphosphate monopotassium salt dihydrate	Merck	A5285	store at -20 °C
Antimycin A	Merck	A8674	store at -20 °C
Bovine serum albumin	Merck	A3803	store at 2 - 8 °C
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone	Merck	C2920	store at -20 °C
Dimethyl sulfoxide	Merck	D8418	store at RT
D-Mannitol	Merck	63559	store at RT
Dulbecco's phosphate buffered saline	Gibco	14190-144	store at RT
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid	Merck	03777	store at RT
HEPES	Merck	H7523	store at RT
L(-)Malic acid disodium salt	Merck	M9138	store at RT
L-Glutamic acid sodium salt hydrate	Merck	G5889	store at RT
Magnissium chloride hexahydrate	Merck	M2670	store at RT
Oligomycin	Merck	O4876	store at -20 °C
Palmitoyl-DL-carnitine chloride	Merck	P4509	store at -20 °C
Potassium hydroxide	Merck	484016	store at RT
Potassium phosphate monobasic	Merck	P5655	store at RT
Rotenone	Merck	R8875	store at -20 °C
Seahorse Wave Desktop Software	Agilent technologies		Download from www.agilent.com
Seahorse XFe96 Analyzer	Agilent technologies
Seahorse XFe96 FluxPak	Agilent technologies	102416-100	XFe96 sensor cartridges and XF96 cell culture microplates
Sodium pyruvate	Merck	P2256	store at 2 - 8 °C
Sodium succinate dibasic hexahydrate	Merck	S2378	store at RT
Sucrose	Merck	S7903	store at RT
Water	Merck	W3500	store at RT
XF calibrant	Agilent technologies	100840-000	store at RT
XF Plasma membrane permeabilizer	Agilent technologies	102504-100	Recombinant perfringolysin O (rPFO) - Aliquot and store at -20 °C