Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.
Most of the cell's energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.
Mitochondria are the primary metabolic organelles in the cell1. These specialized membrane-enclosed organelles use nutrient molecules to produce energy in the form of adenosine triphosphate (ATP) by OXPHOS. This process relies on the transfer of electrons from donor molecules in a series of redox reactions in the ETC2. CoQ is the only redox-active lipid that is endogenously produced in all cellular membranes and circulating lipoproteins that shows antioxidant function3. It is an essential component of the ETC, transferring electrons from NADH-dependent complex I and FADH2-dependent complex II to complex III, although many other reductases can drive the reduction of mitochondrial CoQ to ubiquinol as a mandatory step in multiple cellular metabolic pathways4,5.
Throughout the process, an electrochemical proton gradient is created across the mitochondrial inner membrane, which is transformed into biologically active energy by the ATP synthase complex V2. Consequently, mitochondrial dysfunction leads to a myriad of pathological conditions mainly affecting tissues with high-energy requirements-the brain, heart, and skeletal muscle6,7. Therefore, it is fundamental to develop methods to accurately analyze mitochondrial bioenergetics to investigate its role in health and disease, particularly in highly energetic tissues such as skeletal muscles.
The Clark-type oxygen electrode has been classically used in the study of mitochondrial respiration8. However, this system has been progressively displaced by higher-resolution technologies, with microplate-based oxygen consumption technologies such as Agilent Seahorse XF analyzers being especially popular9. In the skeletal muscle field, these studies are typically conducted in cultured cells, mainly in the C2C12 immortalized mouse myoblast cell line or primary cultures derived from satellite cells10,11. However, these studies do not fully recapitulate the situation in vivo, especially when investigating mitochondrial biology and function at the tissue level upon specific insults, nongenetic interventions, or genetic manipulations.
Furthermore, respiration assays in cells are more complex due to additional factors, including extra-mitochondrial demand of ATP and assay substrates or signaling events, which could mislead the interpretation of the results. Alternatively, it is also possible to use single or bundles of freshly isolated myofibers from muscles. However, the isolation method is technically challenging and only feasible for a few muscle types. In this case, flexor digitorum brevis (FDB) and extensor digitorum longus (EDL) muscles are mainly used10,12,13, although a few reports describe the use of other muscle types as well14,15.
Bioenergetic profiling of skeletal muscle sections has also been reported16. The major advantage of this method is that intact muscles can be studied (the authors show that slicing through fibers does not disturb results when compared with isolated myofibers). However, mitochondrial access to substrates and assay inhibitors is limited, and thus, only a few parameters can be measured16. Finally, isolated mitochondria can be likewise employed9,17,18,19. In this case, mitochondria lose their cytosolic environment, which could affect their function. In contrast, this method guarantees access to substrates and inhibitors, enables the analysis of a plethora of sample types, and typically requires less material.
This paper describes a method to perform the bioenergetic profiling of isolated mitochondria from mouse skeletal muscle using microplate-based respirometric assays (Figure 1). In particular, three protocols are detailed: the Coupling Assay, CA to assess the degree of coupling between the ETC and the OXPHOS machinery; the Electron Flow Assay, EFA to measure the activity of the individual ETC complexes; and the BOX assay to determine mitochondrial β-oxidation capacity. Notably, only small amounts of samples are required compared with conventional respirometry methods. The isolation protocol used here has been modified from the method published elsewhere18.
Mouse housing and tissue collection were performed using protocols approved by the Universidad Pablo de Olavide Ethics Committee (Sevilla, Spain; protocols 24/04/2018/056 and 12/03/2021/033) in accordance with Spanish Royal Decree 53/2013, European Directive 2010/63/EU, and other relevant guidelines.
1. Preparation of stocks, buffers, and reagents for the respiration assays
2. Muscle dissection, homogenization, and mitochondrial isolation
3. Preparation of the microplate-based respirometric assays
4. Analysis of the results
The protocol presented here allows the in vivo analysis of mitochondrial respiration through the isolation of mitochondria from mouse skeletal muscle. An outline of the method is shown in Figure 1. After dissecting skeletal muscles from the hindlimbs (Figure 2), tissues are homogenized and mitochondria purified, under isotonic conditions, through serial centrifugations. The purity of the different fractions obtained during the isolation process can be analyzed by western blot using antibodies against different organelle markers. Figure 3A shows the general protein profile from the different fractions showing distinct protein populations in the fractions isolated.
Figure 3B shows that all mitochondrial content is in the mitochondrial fraction, as evidenced by the strong signals for markers of the outer (VDAC and TOMM20) and inner (TIMM23) mitochondrial membranes, and even for nucleoid-associated proteins (mtTFA). All nuclei and cytoskeleton (β-actin) are present in Fraction N, which represents nuclei and undisrupted material (Figure 3C). Moreover, most of the endoplasmic reticulum (ER) (Calnexin), plasma membrane (Na+/K+-ATPaseα1), or cytoplasm markers (LDHA, HSP70, or AKT) remain in the SN1 or SN2 fractions, highlighting the high purity obtained during isolation (Figure 3C). However, there are a few traces of ER contamination in the mitochondrial fraction, possibly due to the proximity of these organelles. Given the results obtained in subsequent respiratory assays, the isolation procedure described here yields highly pure, viable mitochondrial preparations.
Both CA and BOX assays permit the calculation of different mitochondrial states in the presence of several substrates that stimulate specific metabolic pathways. Specifically, these assays are performed in the presence of pyruvic acid or palmitoyl-L-carnitine chloride. Pyruvic acid is a substrate of the Krebs cycle; malic acid is a Krebs cycle intermediary and an NADH inductor, while palmitoyl-L-carnitine is a substrate of the fatty acid β-oxidation pathway. (Figure 4A,B). In both assays, the first state that can be measured is the basal respiration rate, which represents mitochondrial respiration in the presence of the substrates initially added to the assay medium. Then, mitochondrial State III is achieved, which represents ATP production from ADP and inorganic phosphate, showing the maximal respiration in a coupled state. Thus, there is an increase in oxygen consumption after ADP addition, as observed in Figure 4A,B.
Further, State IVo indicates the proton leak associated with the inhibition of ATP synthase by oligomycin, which leads to a decrease in respiration, as observed in the CA and BOX graphs (Figure 4A,B). The addition of FCCP leads to State IIIu, which shows the maximal respiratory capacity that mitochondria can display in an uncoupled state when oxygen consumption reaches its highest level in these assays. To calculate all these states, it is fundamental first to determine nonmitochondrial respiration, which is obtained at the end of the assay when Antimycin A and rotenone are injected to inhibit oxidative respiration. As shown in Figure 4A,B, these values need to be always below basal respiration and very close to zero in the case of assays with isolated mitochondria like these assays described here. Finally, the RCR parameter must be calculated to assess mitochondrial integrity. It reflects whether mitochondria can react to ADP by producing ATP at a high rate with a low proton leak. The mean RCR values found in the CA and BOX assays were 5.78 ± 1.03 and 3.47 ± 0.42 (Figure 4A,B), respectively, which are in agreement with optimal RCRs reported previously21,22,23,24.
Additionally, the EFA examines the activity of the ETC complexes individually or in combination through the injection of specific substrates and inhibitors (Figure 4C). CI-CIV activity represents mitochondrial respiration based on pyruvate and malate substrates when no complexes are inhibited; in this case, most of the electrons go through CI. As rotenone is a specific inhibitor of CI, CI is blocked after its addition, resulting in decreased oxygen consumption (Figure 4C). CII-CIII-CIV activity shows respiration when only CI is blocked, and CII is activated: succinate, injected through port B, is a specific substrate of complex II (succinate dehydrogenase). Thus, CII is reduced, initiating the electron flow from CII to CIV, while CI is still inhibited by the previous rotenone injection, producing an increase in oxygen consumption (Figure 4C). Antimycin A addition inhibits CIII, blocking the electron flow through the ETC and reducing oxygen consumption. Further, CIV activity is determined when it is stimulated by cytochrome C oxidation after being reduced by the addition of ascorbic acid/TMPD; Figure 4C shows that CIV activation produces an increase in oxygen consumption. Finally, residual activity refers to oxygen consumption when the oxidative phosphorylation chain is inactivated after rotenone and Antimycin A addition. This value establishes the background for the calculation of the activity of the complexes as indicated in protocol steps 2.2-2.4 in section 4.
Figure 1: Schematic visualization of the method. Abbreviations: OCR = oxygen consumption rate; ADP = adenosine diphosphate; OL = oligomycin; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; AntA = Antimycin A; Rot = rotenone. Please click here to view a larger version of this figure.
Figure 2: Dissection and isolation of murine skeletal muscles from hindlimbs for mitochondrial respiration analyses. (A) The right hindlimb was stretched and immobilized with tape. (B) An incision was made through the skin from next to the ankle, stopping proximal to the knee. Connective tissue overlying the TA was carefully removed. EDL (C) and TA (D) tendons were sectioned close to their insertions. The TA tendon was pulled up to ease the muscle away from the underlying musculature and bone. Then, the TA was cut proximally, close to the ventral crest to the tibia. Similarly, the EDL muscle was eased away, and the proximal tendon was meticulously cut at the side of the knee. (F) Right hindlimb after TA and EDL dissection. (G) In the posterior hindlimb, the Achilles tendon was sectioned to dissect GA and soleus muscles. (H) GA and soleus were carefully liberated from the tibial bone. (I) The remaining muscles were collected from ankle to knee until only the fibula and tibial bones remained. (J) Hindlimb after dissection. (K) Quadriceps was dissected. (L) All skeletal muscles collected for posterior mitochondrial isolation. The process was performed for both hindlimbs. Abbreviations: TA = tibialis anterior; EDL = extensor digitorum longus; GA = gastrocnemius. Please click here to view a larger version of this figure.
Figure 3: Purity of the fragmentation process. (A) General protein profile stained with Coomassie blue. (B) Mitochondrial protein purity profile. (C) Nonmitochondrial markers. The different fractions obtained during mitochondria isolation were loaded and incubated with antibodies against different proteins in specific subcellular fractions. Abbreviations: fraction N = non-homogenized tissue and nuclei; SN1 = supernatant number 1: cytoplasm + organelles; SN2 = supernatant number 2: cytoplasm + organelles; VDAC = voltage-dependent anion channel; TOMM20 = translocase of outer mitochondrial membrane 20; mtTFA = mitochondrial transcription factor A; TIMM23 = translocase of inner mitochondrial membrane 23; LDHA = lactate dehydrogenase A; HSP70 = heat shock protein 70; ER = endoplasmic reticulum. Please click here to view a larger version of this figure.
Figure 4: Representative results. Bioenergetic profiling of mitochondria isolated from hindlimb muscles of a 13-month-old male wild type C57BL/6N mouse. The injection points of the different substrates and inhibitors are indicated in each assay. The performance of the ETC and OXPHOS machinery can be analyzed using pyruvate and malate, or palmitoyl-L-carnitine and malate as substrates in the Coupling Assay (A) or β-oxidation of FA assays (B), respectively. (C) The electron flow assay allows the study of individual mitochondrial complexes in the presence of pyruvate, malate, and FCCP; 10 µg of mitochondria were plated per well. Results are displayed following the middle-point representation option to visualize results in an aggregated form. This is in contrast to the point-to-point option, which would enable the visualization of the 9 consecutive measurements usually performed during each measurement step. The type of visualization can be changed after completing the assay under the Kinetic line chart options in the analysis software. Abbreviations: FA = fatty acids; OCR = oxygen consumption rate; ADP = adenosine diphosphate; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; RCR = respiratory control ratio; EFA = Electron Flow Assay; BOX = β-oxidation of FA. Please click here to view a larger version of this figure.
Coupling Assay | ||||||
Port | Inhibitor | [Stock] | [Injection Port] | [Final] | Recipe | |
Inhib | CAM-BSA | |||||
A | ADP | 100 mM | 50 mM | 5 mM | 650 μL | 650 μL |
B | Oligomycin | 4 mM | 31.6 μM | 3.16 μM | 11.3 μL | 1418.7 μL |
C | FCCP | 20 mM | 60 μM | 6 μM | 4.68 μL | 1555.32 μL |
D | Antimycin A // Rotenone | 5 mM // 4 mM | 40 μM // 50 μM | 4 μM // 5 μM | 13.52 μL // 21.12 μL | 1655.36 μL |
Electron Flow Assay | ||||||
Port | Inhibitor | [Stock] | [Injection Port] | [Final] | Recipe | |
Inhib | EFAM-BSA | |||||
A | Rotenone | 4 mM | 50 μM | 5 μM | 16.25 μL | 1283.7 μL |
B | Succinate | 500 mM | 100 mM | 10 mM | 286 μL | 1144 μL |
C | Antimycin A | 5mM | 4 μM | 4 μM | 12.48 μL | 1547.5 μL |
D | Ascorbate // TMPD | 1 M // 50 mM | 100 mM // 1 mM | 10 mM // 100 μM | 169 μL // 33.8 μL | 1487.2 μL |
β-Oxidation Assay | ||||||
Port | Inhibitor | [Stock] | [Injection Port] | [Final] | Recipe | |
Inhib | BOX-BSA | |||||
A | ADP | 100 mM | 40 mM | 4 mM | 520 μL | 780 μL |
B | Oligomycin | 4 mM | 31.6 μM | 3.16 μM | 11.3 μL | 1418.7 μL |
C | FCCP | 20 mM | 60 μM | 6 μM | 4.68 μL | 1555.32 μL |
D | Antimycin A // Rotenone | 5 mM // 4 mM | 40 μM // 20 μM | 4 μM // 2 μM | 13.52 μL // 8.45 μL | 1668 μL |
Table 1: Preparation of inhibitor solutions for the different assays. The [Stock] column shows the concentrations of the stock solutions of the compound indicated in the Inhibitor column. [Injection Port] refers to the concentration of the inhibitor solutions to be loaded in the different ports of the cartridge, while the [Final] column indicates the final concentration of the inhibitors in the wells. Finally, the Recipe columns specify the volumes of stock inhibitor solutions and BSA-free media that must be mixed to prepare the solutions to be loaded in the injection ports. Abbreviations: ADP = adenosine diphosphate; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; TMPD = N,N,N′,N′-tetramethyl-para-phenylene-diamine; Inhib = inhibitor; BSA = bovine serum albumin; CAM-BSA = BSA-free coupling assay medium; EFAM-BSA = BSA-free electron flow assay medium; BOX-BSA = BSA-free β-oxidation of fatty acid assay medium.
Port | Volume injected | Stock prepared (26 wells) |
A | 50 μL | 1300 μL |
B | 55 μL | 1430 μL |
C | 60 μL | 1560 μL |
D | 65 μL | 1690 μL |
Table 2: Calculation of injection volumes.
Action | Time (minutes) | Repetition | Port |
Calibrate | 15-20 | ||
Wait | 10 | ||
Mix + Wait | 1 + 3 | × 2 | |
Mix | 1 | ||
Measure + Mix | 3 + 1 | × 2 | |
Inject | A | ||
Mix | 1 | ||
Measure | 3 | ||
Mix | 1 | ||
Inject | B | ||
Mix | 1 | ||
Measure | 3 | ||
Mix | 1 | ||
Inject | C | ||
Mix | 1 | ||
Measure | 3 | ||
Mix | 1 | ||
Inject | D | ||
Mix + Measure | 1 + 3 | × 2 |
Table 3: Protocol settings for the different assays.
All methods used to study mitochondrial respiration have their limitations; hence, it is crucial to select the method that best suits a specific experimental question. This work provides an updated and detailed protocol to isolate mitochondria from mouse skeletal muscle to perform different respiratory assays to investigate mitochondrial function. Indeed, the study of mitochondrial bioenergetics in isolated mitochondria using microplate-based technologies is valuable to study tissue-specific respiration in terms of reproducibility, reliability, and complexity. Further, the use of isolated mitochondria confers control over substrate availability, facilitating the understanding of the underlying biological processes. Another positive aspect of using isolated mitochondria is that it is possible to study State III because the ATP generated after ADP injection is not converted again into new ADP as most of the transmembrane ATPases are lost. Moreover, in the assays described here, oligomycin is added to block potential nonspecific ATP synthesis20.
Several important considerations need to be highlighted for this protocol. First, isolated mitochondria are extremely sensitive and should be handled with care. The high content of sucrose and mannitol in the buffers used ensures the optimal osmotic conditions to protect the isolated mitochondria from damage. Nevertheless, the assay duration should be kept at a minimum to preserve mitochondrial viability and prevent detachment from the microplate once seeded. Thus, given that protocol steps, such as the preparation of assay media and substrate and inhibitor solutions, or the loading of the inhibitors in the respirometric assay cartridge, are time-consuming, every step should be perfectly coordinated. Further, unlike other methods based on enzymatic digestion21,24 and mortar and pestle homogenization17,19,21,23,24, the method described here relies on the use of an automated homogenizer, enabling fast and efficient sample homogenization. Importantly, mitochondrial suspensions are less sensitive if highly concentrated. Thus, only prepare dilutions immediately before using them. Finally, it is essential to work quickly during the respirometric assay proper. Usually, when analyzing cells in culture in these assays, three consecutive measurement steps are performed after the injection of substrates or inhibitors, which results in protocols that extend between 40 and 50 min., To ensure the viability of the isolated mitochondria throughout the whole procedure, the number of measurement steps is often kept to a minimum due to their vulnerability19,23,25, reducing the time it takes to complete the respirometric analysis to ~20 min. The protocols described in this work (Table 3) follow this trend, even though other previously published methods report efficient OCR profiles with longer and more standard measurement steps26.
For example, if two consecutive assays are going to be run with the same set of samples on the same day, prepare all reagents for the second assay during the first one except for the mitochondrial dilutions: only dilute, seed, and spin mitochondria immediately before starting the second assay, while the instrument is being calibrated. Once the experiment is completed, the RCR parameter (section 4, step 1.7) must be calculated to assess the quality of the mitochondrial preparations used. Usually, RCR values between 3.5 and 5 denote that mitochondrial integrity is optimal and show functional coupled oxidative respiration25. Assays with lower RCR values must be discarded because mitochondrial integrity was compromised during isolation. In these cases, consider washing the mitochondrial pellet twice (section 2, steps 19-21)22, as previously reported.
Second, maintaining the correct pH throughout the procedure is critical to obtaining successful results. Ensure that the pH of all solutions is set as indicated, that is, pH 7.2, and that the pH is always adjusted with KOH unless otherwise stated. Notably, the pH of BSA-containing buffers should not be directly measured with the pH meter as BSA can damage the probe. Thus, adjust the pH in the corresponding buffers before adding BSA. To avoid changes in the pH due to BSA addition, always use FFA BSA. Further, some ultrapure H2O batches could be acidic, so the use of commercially purified, pH 7 H2O is recommended. Furthermore, it is necessary to ensure that the pH meter is correctly calibrated and that it is the appropriate model to measure the pH of the chemicals to be prepared.
Third, it is crucial to correctly perform the sample collection step for protein measurement of the final mitochondrial suspension (section 2, step 21) to prevent mitochondrial destruction. Resuspend the pellet quickly but thoroughly, collect an aliquot, and add FFA BSA to the remaining suspension to conserve the osmotic balance and protect the mitochondria from damage. If FFA BSA were already present in the resuspension buffer, and thus, during protein quantification, the high protein content would confound the quantification results. This is why FFA BSA needs to be added after setting an aliquot aside for quantification.
Fourth, this method is highly versatile and can be adjusted to the experimenter's needs. For example, if the animal model under study is characterized by reduced muscle mass (e.g., sarcopenic mice), additional muscles can increase mitochondrial yield. Although an adult wild-type male C57BL/6N mouse was used in this work, animals of any gender, age, or genetic background, as well as specific muscle types, can be employed instead.
Fifth, respiratory values can differ between experiments due to slight variations in the composition of buffers and reagents, which need to be prepared fresh every time. Age, sex, genetic background, or environmental conditions can also have a profound impact on the results. However, a successful experiment should present increases in oxygen consumption after the injection of complex substrates such as ADP and succinate, decreases after the injection of inhibitors such as oligomycin or rotenone, and marked increases when the potent uncoupler FCCP is added. Moreover, to prevent high technical variability, ensure thorough resuspension of mitochondrial pellets to obtain homogeneous suspensions before seeding them in the microplate.
Sixth, if basal respiratory values are low, consider performing a titration experiment to adjust the amount of protein for seeding. Finally, because this work provides a method to obtain crude mitochondrial extracts, a certain grade of impurity associated with other organelles, mainly ER, is expected. A more aggressive fragmentation process to remove these impurities would be detrimental to mitochondrial viability, hindering the performance of respiratory assays. If purity is a concern, especially if it is not consistent between samples, assay results can be normalized relative to mitochondrial purity after running western blots against different organelle markers (Figure 3). To our knowledge, this is the first method that highlights concerns about mitochondrial purity in crude extracts. Usually, in other protocols for respirometric assays with isolated mitochondria, the same amount of protein extract is seeded in the wells to compare samples, assuming that the same amount of mitochondria is used in all cases. This is a reasonable assumption considering that isolation is performed in parallel in all samples. However, the genetic or environmental background of the samples under study may lead to considerable differences in the purity of the mitochondrial extracts. For example, if a given genetic mutation causes increased development of cytoplasmic ER, crude mitochondrial extracts from these animals could contain greater than expected ER content, and consequently, lower than expected mitochondrial protein. Thus, even if the same amount of total protein from control and mutant samples are seeded in the microplate, oxygen consumption of the mutants would be underestimated. Hence, it is recommended that the purity of the extracts of all the conditions under study must be determined. If the values are comparable, no normalization is needed. However, if purity values among samples differ considerably beyond the statistical error the experimenter is willing to accept, they should be used to normalize oxygen consumption results.
The present protocol has some limitations. Of note, this method has been optimized for skeletal muscle tissue isolated from mice. Adjustments may need to be made if using different tissues or skeletal muscle from different species. Additionally, when analyses are performed on isolated mitochondria, the normal cellular microenvironment is lost due to the absence of cellular context and the other organelles that could interact with mitochondria. This could lead to results that slightly deviate from physiological conditions. Finally, as the mitochondria are centrifuged, they adhere to the bottom of the wells. While completely necessary, the potential effects of centrifugation on their normal state are unknown.
It is important to take into account that, after performing the respirometric assay analyses, a reduction in oxygen consumption associated with ATP-linked or FCCP-stimulated respiration in isolated mitochondria could indicate potential mitochondrial alterations that could be explained by20: 1) restricted transport of substrates across the inner mitochondrial membrane, 2) decreased activity of the enzymes involved in the rate-limiting reactions of specific metabolic pathways such as the ETC, Krebs cycle, or β-oxidation, or 3) impaired ETC function, among other possible explanations.
In summary, the updated method described here represents an adequate and reliable way to assess ETC and OXPHOS function from skeletal muscle tissue. It has multiple applications to evaluate how genetic modifications or the environment could affect mitochondrial respiration contributing to a specific phenotype. Remarkably, the present protocol could be adapted to other model organisms or be used with human samples to evaluate potential mitochondrial dysfunctions associated with human disease.
The authors have nothing to disclose.
We wish to thank Juan J. Tena for the use of the homogenizer and the CABD Proteomics and Animal Husbandry facilities for technical support. This work was supported by the Spanish Ministry of Education, Culture and Sports through fellowship FPU16/03264 to J.D.H.C., the Association Française contre les Myopathies (AFM) through fellowship grant #22450 to C.V.-G., an Institutional Grant MDM-2016-0687 (Maria de Maeztu Excellence Unit, Department of Gene Regulation and Morphogenesis at CABD) and BFU2017-83150-P to J.J.C. The Junta de Andalucía grant P18-RT-4572, the FEDER Funding Program from the European Union, and Spanish Ministry of Science, Innovation and Universities grant RED2018-102576-T to P.N.
ADP | Sigma | A5285 | Stock at -20 °C |
AKT antibody | Cell Signaling Technology | C67E7 | Rabbit (Host species) |
anti-Goat HRP | Sigma | 401504 | Rabbit (Host species) |
anti-Mouse HRP | Cell Signaling | #7076 | Horse (Host species) |
Antimycin A | Sigma | A8674 | Stock at -20 °C |
anti-Rabbit HRP | Cell Signaling | #7074 | Goat (Host species) |
Ascorbic acid | Sigma | A5960 | Stock at RT |
Bactin antibody | Sigma | MBS4-48085 | Goat (Host species) |
Bio-Rad Protein Assay Kit II | Bio-Rad | 5000002 | It includes 5x Bradford reagent and BSA of known concentration for the standard curve |
BSA, fraction V, Fatty Acid-Free | Calbiochem | 126575 | Stock at 4 °C |
C tube | Miltenyi Biotec | 130-093-237 | Purple lid |
Calnexin antibody | ThermoFisher | MA3-027 | Mouse (Host species) |
D-mannitol | Sigma | M4125 | Stock at RT |
EDTA | BDH | 280254D | Stock at 4 °C |
EGTA | Sigma | E-4378 | Stock at RT |
FCCP | Sigma | C2920 | Stock at -20 °C |
gentleMACS Dissociator | Miltenyi Biotec | 130-093-235 | Homogenizer |
HEPES | Sigma | H3375 | Stock at RT |
HSP70 antibody | Proteintech | 10995-1-AP | Rabbit (Host species) |
LDH-A antibody | Santa Cruz Biotechnology | SC27230 | Goat (Host species) |
Magnesium chloride | ChemCruz | sc-255260A | Stock at RT |
Malic acid | Sigma | P1645 | Stock at RT |
Microplate spectrophotometer | BMG LABTECH GmbH | POLARstar OMEGA S/N 415-0292 | Stock at RT |
Milli-Q water | Millipore system | F7HA17757A | Ultrapure water |
mtTFA antibody | Santa Cruz Biotechnology | SC23588 | Goat (Host species) |
Na+/K+-ATPase α1 antibody | Novus Biologicals | NB300-14755 | Mouse (Host species) |
Oligomycin | Sigma | O4876 | Stock at -20 °C |
Palmitoyl-L-carnitine | Sigma | P1645 | Stock at -20 °C |
PBS tablets | Sigma | P4417-100TAB | 1x stock at RT |
Potassium dihydrogen phosphate | ChemCruz | sc-203211 | Stock at RT |
Potassium hydroxide | Sigma | 60377 | Stock at RT |
Pyruvic acid | Sigma | 107360 | Stock at 4 °C |
Rotenone | Sigma | R8875 | Stock at -20 °C |
Seahorse XF24 mitochondrial flux analyzer | Agilent Technologies | 420179 | XFe24 model |
Seahorse XFe24 FluxPak mini | Agilent Technologies | 102342-100 | The kit includes cartridges, microplates, and calibrant solution |
Succinate | Sigma | S7626 | Stock at RT |
Sucrose | Sigma | S9378 | Stock at RT |
TIMM23 antibody | Abcam | ab230253 | Rabbit (Host species) |
TMPD | Sigma | T7394 | Stock at -20 °C |
TOMM20 antibody | Abcam | ab56783 | Mouse (Host species) |
VDAC antibody | Abcam | ab15895 | Rabbit (Host species) |