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 continuous flux of substrates is essential for the flow of the citric acid cycle to generate high energy 'electron donors', which deliver electrons to the electron transport chain that generates a proton gradient across the inner mitochondrial membrane, enabling ATP-synthase to phosphorylate ADP to ATP⁴. Therefore, an experimental design to assay mitochondrial respiration must include the sample nature (intact cells, permeabilized cells, or isolated mitochondria) and mitochondrial substrates.

Cells keep a store of indigenous substrates⁵, and mitochondria oxidize several types of substrates simultaneously⁶, which complicates the interpretation of results obtained from experiments performed on intact cells. A common approach to investigate mitochondrial ability to oxidize a selected substrate is to isolate mitochondria or permeabilize the investigated cells⁵. Although isolated mitochondria are ideal for quantitative studies, the isolation process is laborious. It faces technical difficulties such as the need for large sample size, purity of the yield, and reproducibility of the technique⁵. Permeabilized cells offer a solution for the disadvantages of mitochondrial isolation; however, routine permeabilizing agents of detergent nature are not specific and may damage mitochondrial membranes⁵.

Recombinant perfringolysin O (rPFO) was offered as a selective plasma membrane permeabilizing agent⁷, and it was used successfully in combination with an extracellular flux analyzer in several studies^7,8,9,10. We have modified a protocol using rPFO to screen mitochondrial substrate flux using XFe96 extracellular flux analyzer. In this protocol, four different substrate oxidizing pathways in two cellular phenotypes are compared while having sufficient replicates and the proper control for each tested material.

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. Adjust the pH to 7.4 with 5 N KOH, and then add water to bring the volume up to 100 mL. Filter-sterilize and store the aliquots at -20 °C until the measurement day.
   3. Mitochondrial substrates: Prepare 1 M stock solutions of sodium succinate, sodium pyruvate, and sodium glutamate in sterile water. Prepare 100 mM stock solution of sodium malate in sterile water and use prewarmed sterile water to prepare a 10 mM stock solution of palmitoyl carnitine. Adjust pH of each solution to 7.4 by 5 N KOH and filter-sterilize. Store the substrates at 2-8 °C. At the time of use, warm palmitoyl carnitine to 37 °C to dissolve any precipitates. For later use, store aliquots of all the substrates except pyruvate at -20 °C.
   4. Mitochondrial inhibitors: Use dimethyl sulfoxide (DMSO) to prepare stock solutions of 25 mM oligomycin, 50 mM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 20 mM rotenone, and 20 mM antimycin A. Store the aliquots at -20 °C.
2. Seeding and treating the cells: As shown in Figure 2, seed the cells in columns 2-11. Columns 1 and 12 must be left empty to serve as background wells. In this work, HepG2 and A549 cells were used. 
   1. Seed the cells at a density of 20,000 cells per well. Use 50-80 µL of cell culture medium for seeding.
   2. Fill the blank wells with an equal volume of cell culture medium and incubate the cells at 37 °C in a humidified incubator with 5% CO₂. Allow the cells to attach for 3-4 hours, and then add 100 µL of cell culture medium to all wells.
   3. With that final medium addition, apply the treatment in columns 7–11. In this work, the experimental group was treated with 1 mM metformin hydrochloride for 16 hours, and the control group was treated with an equal volume of sterile distilled water as a vehicle control.
3. Hydrating the sensors: Pipette 200 µL of sterile water per well into the utility plate, then carefully return the sensor cartridge while immersing the sensors in water. Incubate the cartridge in a CO₂-free incubator at 37 °C till the next day.
4. The assay protocol template: Switch on the analyzer (see Table of Materials) and the controller unit. Start the instrument control and data acquisition software and design the assay protocol as described in Table 2. Under Group Definitions, create four injection strategies where Port A differs according to the injected substrate (Figure 3). Name the strategies after the substrates or their abbreviations.
5. To ports B, C, and D, assign the compounds oligomycin, FCCP, and rotenone/antimycin A, respectively. Create eight groups and name them as shown in Figure 4. Under Plate Map assign the groups to the corresponding wells, then save the protocol as a ready-to-use template. Leave the analyzer switched on to allow the temperature to stabilize overnight. Keep the analyzer in a place with a stable temperature to avoid sudden temperature changes.

2. The day of the assay

1. Replacing water with the calibrant: Discard the water from the utility plate and pipette 200 µL of prewarmed calibrant per well into the utility plate. Return the cartridge to the CO₂-free incubator until the time of the assay. To avoid rapid evaporation of the calibrant, maintain a source of humidity inside the incubator and turn off or reduce the fan speed to a minimum.
2. Preparing the working solutions: Start by warming 2x MAS, 5% BSA, and sterile water to 37 °C. Meanwhile, allow the inhibitor stocks to reach room temperature. Use warm 2x MAS and sterile distilled water to prepare 5 mL of the working concentration of the substrates and inhibitors as described in Table 3.
3. Loading the injection ports: As shown in Figure 3, load 20 µL of the substrates into port A. Load succinate/rotenone mixture into port A of rows A and B. Load pyruvate/malate mixture into port A of rows C and D. Load glutamate/malate mixture into port A of rows E and F. Load palmitoyl carnitine/malate mixture into port A of rows G and H. For the whole plate, load port B with 22 µL of oligomycin preparation, port C with 25 µL of FCCP preparation, and port D with 27 µL of rotenone/antimycin A mixture.
4. Starting the calibration step: Under Run Assay tab, click on Start Run to start the assay. Insert the loaded sensor cartridge and start the calibration step. Wait for the calibration to complete before proceeding to the next step.
5. Preparation of the assay medium (MAS-BSA-rPFO): To prepare 20 mL of the assay medium, mix 10 mL of 2x MAS, 9.2 mL of sterile water, and 0.8 mL of 5% BSA in a 50 mL tube. Add 2 µL of 10 µM rPFO to attain a concentration of 1 nM and resuspend the mixture with gentle pipetting. Avoid shaking and do not use a vortex mixer for mixing. Incubate the tube at 37 °C until the time of use.
6. Washing the cells: Wash the cells and the empty blank wells two times with prewarmed calcium- and magnesium-free phosphate buffered saline (PBS) using a multichannel pipette. Avoid reusing the same tips to discard the cell culture medium and to add PBS. Perform this step outside the laminar flow to protect the cells from drying out by the airflow.
7. Cell permeabilization in assay medium: Using a multichannel pipette, discard the PBS and replace it with 180 µL of the prewarmed assay medium (MAS-BSA-rPFO).
8. Starting the measurement: Immediately after permeabilization, replace the utility plate of the calibrated sensor cartridge with the cell plate and start the measurement.

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

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenosine 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
Magnesium 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