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* These authors contributed equally
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.
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.
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....
1. One day before the assay
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.......
This protocol is a modification of previously published studies7,8,9,10 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 .......
The authors thank the staff members of the Department of Physiology in the Faculty of Medicine in Hradec Králové and the Department of Pathophysiology in the Third Faculty of Medicine for the help with chemicals and samples preparation. This work was supported by Charles University grant programs PROGRES Q40/02, Czech Ministry of Health grant NU21-01-00259, Czech science foundation grant 18-10144 and INOMED project CZ.02.1.01/0.0/0.0/18_069/0010046 funded by the Ministry of Education, Youth and Sports of the Czech Republic and by the European Union.
....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 |
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