Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Summary

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.

Abstract

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.

Introduction

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 pathways^4,5.

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 muscle^6,7. 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 cells^10,11. 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 used^10,12,13, although a few reports describe the use of other muscle types as well^14,15.

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 employed^9,17,18,19. 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.

Protocol

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

1. Prepare the following stock solutions, which can be stored at the indicated temperature for months. Use ultrapure H₂O in all cases.
   1. Dissolve phosphate-buffered saline (PBS) tablets in H₂O (1 tablet per 200 mL of H₂O) to prepare 1x PBS. Autoclave the solution and store it at room temperature (RT).
   2. Dissolve 2 g of NaOH pellets in 50 mL of H₂O to prepare 1 M NaOH.
   3. Prepare 0.1 M, 1 M, 5 M, and 10 M KOH stocks for pH calibration.
   4. Add 14.612 g of EDTA to 70 mL of H₂O. Add NaOH pellets until the pH reaches 8.0 so that EDTA fully dissolves. Add H₂O quantum satis (QS) to 100 mL. Autoclave the resulting 0.5 M EDTA (pH 8) solution and store it at RT.
   5. Add 19 g EGTA to 70 mL of H₂O. Add NaOH pellets until the pH reaches 8.0 to allow the EGTA to dissolve fully. Add H₂O QS to 100 mL. Autoclave the resulting 0.5 M EGTA (pH 8) solution and store it at RT.
   6. Add 2 mL of 0.5 M EDTA to 98 mL of PBS. Store the resulting 10 mM EDTA/PBS solution at RT.
   7. Dissolve 5.958 g of HEPES in 40 mL H₂O. Adjust the pH to 7.2 with KOH and QS to 50 mL with H₂O. Filter the resulting 0.5 M HEPES buffer through a 0.45 µm mesh and store it at RT.
   8. Dissolve 3.402 g of KH₂PO₄ in up to 50 mL of H₂O. Filter the resulting 0.5 M KH₂PO₄ solution through a 0.45 µm mesh and store it at RT.
   9. Dissolve 9.521 g of anhydrous MgCl₂ in up to 100 mL of H₂O. Autoclave the resulting 1 M MgCl₂ solution and store it at RT.
      NOTE: As this is an exothermic reaction, proceed with caution and dissolve the MgCl₂ on ice.
2. Prepare substrate and inhibitor stock solutions (see Table 1). Aliquot and store them at -20 °C. Avoid freeze-thaw cycles. As an exception, always prepare pyruvic acid immediately before use.
   1. Prepare in ultrapure H₂O: 0.5 M succinate, 0.5 M pyruvic acid, 0.5 M malic acid, 100 mM ADP, 1 M ascorbic acid, and 50 mM N,N,N′,N′-tetramethyl-para-phenylene-diamine (TMPD) (protect from light).
   2. Prepare in 100% dimethyl sulfoxide (DMSO): 50 mM palmitoyl-L-carnitine, 4 mM rotenone, 20 mM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), 4 mM oligomycin, and 5 mM Antimycin A.
      NOTE: Do not exceed 0.1% final DMSO concentration in the microplate wells. Therefore, prepare highly concentrated stock solutions to stay below this limit.
3. Prepare the buffers for mitochondria isolation and protein quantification freshly on the day of the experiment. Use ultrapure H₂O in all cases and keep all buffers on ice unless otherwise stated.
   NOTE: To save time, all reagents can be weighed and kept in the appropriate containers (e.g., 15 mL tubes) at the right temperature on the previous day.
   1. To prepare 10% free fatty acids (FFA) BSA, thoroughly dissolve 150 mg of FFA BSA in 1.5 mL of H₂O by inversion/rotatory wheel. Do not vortex to avoid foaming.
   2. To prepare 1x Bradford reagent, dilute the 5x commercial stock solution with H₂O and keep it in the dark at RT.
   3. To prepare 8x Mitochondria Buffer (MB), dissolve 4.112 g of sucrose and 763 mg of HEPES in 15 mL of H₂O. Adjust the pH to 7.2, add 1.6 mL of 10% FFA BSA and QS to 20 mL with H₂O.
   4. To prepare 20 mL of Isolation Buffer 1 (IB1) (4 mL used per sample), dissolve 400 µL of 0.5 M EDTA, 784 mg of D-mannitol, and 2.5 mL of 8x MB in 15 mL of H₂O. Adjust the pH to 7.2 and QS with H₂O.
   5. To prepare 5 mL of Isolation Buffer 2 (IB2) (500 µL used per sample), dissolve 30 µL of 0.5 M EGTA, 196 mg of D-mannitol, and 625 µL of 8x MB in 4 mL of H₂O. Adjust the pH to 7.2 and QS with H₂O.
   6. To prepare 5 mL of Resuspension Buffer (RB) (200 µL used per sample), dissolve 120 mg of sucrose, 191.3 mg of D-mannitol, 50 µL of 0.5 M HEPES, and 10 µL of 0.5 M EGTA in 4 mL of H₂O. Adjust the pH to 7.2 and QS with H₂O.
   7. To prepare 2x Mitochondrial Assay Solution-1 (MAS-1), dissolve 1.199 g of sucrose, 2.8 g of mannitol, 1 mL of 0.5 M KH₂PO₄, 250 µL of 1 M MgCl₂, 200 µL of 0.5 M HEPES, and 100 µL of 0.5 M EGTA in 20 mL of H₂O. Adjust the pH to 7.2 and QS to 25 mL with H₂O. Keep in ice for short periods. For longer periods, keep it at 4 °C to avoid precipitation.
   8. To prepare 1x Coupling Assay medium (CAM), prepare two different MAS-1-based buffers: 1) CAM+BSA for the CA assay proper, and 2) CAM-BSA for preparing the assay inhibitors. Always keep the pyruvate:malate ratio at 10:1.
      NOTE: As BSA can clog the injection ports, dilute the inhibitors in BSA-free CAM.
      1. To prepare CAM+BSA, dilute 300 µL of 0.5 M pyruvic acid, 30 µL of 0.5 M malic acid, and 7.5 mL of 2x MAS-1 in 6 mL of H₂O. Adjust the pH to 7.2. Add 300 µL of 10% FFA BSA and QS to 15 mL with H₂O.
      2. To prepare CAM-BSA, dilute 120 µL of 0.5 M pyruvic acid, 12 µL of 0.5 M malic acid, and 3 mL of 2x MAS-1 in 2 mL of H₂O. Adjust the pH to 7.2 and QS to 6 mL with H₂O.
   9. To prepare 1x Electron Flow Assay medium (EFAM), prepare both BSA-containing and BSA-free EFAM buffers.
      1. To prepare EFAM+BSA, dilute 300 µL of 0.5 M pyruvic acid, 60 µL of 0.5 M malic acid, 3 µL of 20 mM FCCP, and 7.5 mL of 2x MAS-1 in 6 mL of H₂O. Adjust the pH to 7.2. Add 300 µL of 10% FFA BSA and QS to 15 mL with H₂O.
      2. To prepare EFAM-BSA, dilute 120 µL of 0.5 M pyruvic acid, 24 µL of 0.5 M malic acid, 1.2 µL of 20 mM FCCP, and 3 mL of 2x MAS-1 in 2 mL of H₂O. Adjust the pH to 7.2 and QS to 6 mL with H₂O.
   10. To prepare 1x β-oxidation medium (BOXM), prepare BSA-containing and BSA-free BOXM solutions.
       1. To prepare BOXM+BSA, dilute 12 µL of 50 mM palmitoyl-L-carnitine, 30 µL of 0.5 M malic acid, and 7.5 mL of 2x MAS-1 in 7 mL of H₂O. Adjust the pH to 7.2. Add 300 µL of 10% FFA BSA and QS to 15 mL with H₂O.
       2. To prepare BOXM-BSA, dilute 4.8 µL of 50 mM palmitoyl-L-carnitine, 12 µL of 0.5 M malic acid, and 3 mL of 2x MAS-1 in 2 mL of H₂O. Adjust the pH to 7.2 and QS to 6 mL with H₂O.

2. Muscle dissection, homogenization, and mitochondrial isolation

1. Place all materials and buffers on ice. Ensure all materials are ice-cold during the entire procedure to protect mitochondria from damage.
2. Place three 50 mL beakers per sample on ice, and add the following solutions: 10 mL of PBS in beaker 1, 10 mL of 10 mM EDTA/PBS in beaker 2, and 4 mL of IB1 in beaker 3.
3. Euthanize the mouse by cervical dislocation. Avoid CO₂ euthanasia as skeletal muscles could become hypoxic, interfering with respiration analyses.
4. Spray the right hindlimb with 70% ethanol to prevent fur from shedding, and tape limb to the cork dissection board. Tape the left forelimb as well.
5. Make an incision with a sterile disposable scalpel through the skin from knee to toe.
6. Grip the skin with toothed forceps at ankle level and cut it with fine scissors around the ankle.
7. Ease the skin away from the underlying musculature with fine-tip tweezers in one hand while pulling it up with toothed forceps in the other hand.
8. Dissect all skeletal muscles from ankle to knee (Figure 2).
   1. Remove all connective tissue over the tibialis anterior (TA) muscle to facilitate muscle extraction using fine tweezers and scissors.
   2. Find the four distal tendons of the EDL muscle and section them close to their insertions in the toes. Locate the TA distal tendon and cut it close to its insertion, always below the ankle.
   3. Carefully pull the TA and EDL tendons above the ankle to liberate the loose ends.
   4. Grip the loose ends of the tendons and ease the muscles away from the rest of the musculature and bones by pulling them up. Use fine tweezers or scissors to facilitate the process. Proceed with care to avoid myofiber contraction.
   5. Cut the proximal tendon of the EDL and the TA muscle as close as possible to the kneecap.
   6. Turn the mouse upside down to proceed with gastrocnemius (GA) and soleus muscle extraction.
   7. Grip the pocket formed between the biceps femoris and the GA with toothed forceps. Use fine scissors to separate these muscles and visualize the proximal GA tendon.
   8. Grip the Achilles tendon with fine forceps and carefully cut it with fine scissors. Release the GA and soleus muscles from the underlying bone by pulling them up through their tendons. Cut the proximal GA tendon liberating it together with the underlying soleus.
   9. Carefully dissect the remaining muscles from tendon to tendon following the same procedure until only bones remain.
   10. Re-pin the mouse in the initial position to dissect the quadriceps muscle.
   11. Discard the adipose tissue over the quadriceps at the proximal side using toothed forceps and fine scissors.
   12. Insert fine tweezers between the quadriceps and the femur and move them in both directions along the femur axis to separate the muscle from the bone.
   13. Grip the distal quadriceps muscle tendons with toothed forceps and cut the tendon with fine scissors as close as possible to the kneecap.
   14. Pull the quadriceps up and liberate it at the proximal insertion with fine scissors.
9. Repeat steps 4-8 from this section with the left hindlimb.
10. Rinse all muscles in Beaker 1 first and then in Beaker 2.
11. Transfer all muscles to Beaker 3 and finely mince all muscles with sharp scissors on ice.
12. Transfer the suspension to a C Tube (purple lid), always keeping it in ice.
13. Tightly close the C Tube and attach it upside down onto the sleeve of the homogenizer. Make sure that the sample material is in the area of the rotator/stator. Select the 1-min program called m_mito_tissue_01.
14. Divide the homogenate into two 2 mL prechilled microcentrifuge tubes and centrifuge at 700 × g for 10 min at 4 °C in a tabletop centrifuge.
15. Transfer the supernatants to new 2 mL prechilled tubes, carefully avoiding fat and non-homogenized tissue. Store the pellets at -80 °C for fragmentation purity determination (fraction N) (Figure 3).
16. Centrifuge the supernatants at 10,500 × g for 10 min at 4 °C.
17. Transfer the supernatants to new 2 mL prechilled tubes and label them as Supernatant Number 1 (SN1). Store them at -80 °C for fragmentation purity determination (Figure 3).
18. Resuspend and combine both pellets in a total volume of 500 µL of IB2 in ice.
19. Centrifuge at 10,500 × g for 10 min at 4°C.
20. Transfer the supernatant to a new 2 mL prechilled tube and label it as Supernatant Number 2 (SN2). Store it at -80 °C for fragmentation purity determination (Figure 3).
21. Resuspend the final mitochondrial pellet in 200 µL of RB. Quickly set aside 10 µL for protein quantification and immediately add 10 µL of 10% FFA BSA to the remaining mitochondrial suspension to prevent damage.
22. Determine protein concentration using the Bradford assay.
    1. For the standard curve, prepare 30 µL of serial dilutions of known concentration of a protein in RB buffer. For example, use 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, and 0 mg/mL of BSA.
    2. Prepare 1:3 and 1:6 serial dilutions of the mitochondrial samples in RB buffer.
    3. In a 96-well flat-bottom plate, first load 2.5 µL of sample/standard per well. Next, add 10 µL of 1 M NaOH to each sample and, finally, 200 µL of 1x Bradford reagent. Mix well, avoiding air bubbles. Check visually that the color of the samples is within the calibration line. Always perform the analysis in triplicate.
    4. Incubate the plates for 5 min at RT in the dark, and read the absorbance at 595 nm in a microplate spectrophotometer.
    5. Calculate the standard curve by plotting the absorbance values (y-axis) versus the corresponding protein concentration of the dilutions prepared in step 22.1 in this section (x-axis).
    6. Calculate the total amount of protein in the mitochondrial samples by extrapolation with the standard curve.

3. Preparation of the microplate-based respirometric assays

1. Hydrate the respirometric assay sensor at least 12 h before the experiment with 1 mL of calibration buffer per well. Incubate at 37 °C (no CO₂).
   NOTE: Hydrated sensors can be used for up to 72 h. The cartridge should be handled carefully during this step: if anything touches the sensors, measurement sensibility could be affected.
2. Prechill the preparative centrifuge with the swinging bucket microplate rotor and the corresponding microplate adapters at 4 °C.
3. Turn on the computer, open the analysis software, and select the desired protocol.
   NOTE: A click is heard when the software connects to the oxygen consumption-measuring instrument. The heating sensor should be green and at 37 °C before the experiment starts.
4. Prepare the inhibitor solutions from the stocks indicated in section 1, step 2 according to the assay to be performed. Prepare enough volume for each solution. See Table 1 and Table 2 for reference.
5. Add the appropriate volume of each inhibitor in each port, insert the cartridge into the oxygen consumption-measuring instrument, and start calibration.
   NOTE: Adding inhibitor to the cartridge and inserting the cartridge into the instrument takes15-20 min. Ensure the correct cartridge components are introduced. The cartridge lid and hydro booster could be discarded while sensor cartridge and utility place are needed.
6. Centrifuge the concentrated mitochondrial suspension of section 2, step 21 at 10,500 × g for 10 min at 4 °C.
7. Resuspend the mitochondrial pellet in 100 µL of 1x CAM+BSA, 1x EFAM+BSA, or 1x BOXM+BSA, depending on the protocol to be performed.
8. Further dilute the concentrated mitochondrial sample to a final 0.2 µg/µL concentration in the corresponding assay medium.
9. Seed 50 µL of the suspension (total 10 µg of protein) per well in a prechilled 24-well microplate on ice. Do not add mitochondria in the background correction wells; only add the corresponding assay medium. Store the remaining mitochondrial suspension at -80 °C for fragmentation purity determination (Figure 3).
10. Spin the microplate in the prechilled preparative centrifuge at 2,000 × g for 20 min at 4 °C. Counterweight to balance accordingly.
11. Warm the remaining assay medium at 37 °C during microplate centrifugation.
12. After centrifugation, leave the microplate on the bench for 5 min to equilibrate. Add 450 µL of warm assay medium for a final volume of 500 µL per well at RT. Do this slowly and carefully, adding the medium to the wall of the wells to avoid detaching mitochondria.
13. Place the microplate immediately in the oxygen consumption-measuring instrument without the lid, and start the protocol (Table 3). Ensure that the first step is a 10 min incubation to allow the microplate to warm.
14. Once the experiment is finished, remove the cartridge and microplate, switch off the instrument, and start the analysis.

4. Analysis of the results

1. In the case of the CA and BOX assays, perform the following analyses:
   1. Record nonmitochondrial O₂ consumption, which corresponds to the mean of the values obtained after Antimycin A and rotenone injection.
      ​NOTE: Antimycin A and rotenone are complex III and I inhibitors, respectively.
   2. Calculate basal respiration by subtracting nonmitochondrial O₂ consumption from basal values (measurement points 1 and 2).
   3. Subtract nonmitochondrial O₂ consumption from the values after the injection of the complex V substrate ADP (injection A) to determine mitochondrial state III.
   4. Subtract nonmitochondrial O₂ consumption from the respiration values post injection of the complex V inhibitor oligomycin (injection B) to obtain mitochondrial state IVo.
   5. Calculate mitochondrial state IIIu by deducting nonmitochondrial O₂ consumption from respiration post FCCP injection (injection C).
      NOTE: FCCP is a potent mitochondrial oxidative phosphorylation uncoupler. ATP synthesis is bypassed in its presence, and the ETC attains maximal activity.
   6. Subtract basal respiration values from the mitochondrial state IIIu values to obtain mitochondrial spare capacity, which is the capacity to generate extra ATP in case of increased energy demand.
   7. Divide mitochondrial state IIIu by state IVo values to obtain the Respiratory Control Ratio (RCR).
      NOTE: Negative or null RCR values indicate that mitochondrial coupling is affected.
2. In reference to EFA, perform the following calculations:
   1. Obtain residual activity by calculating the mean value post rotenone and Antimycin A injection (injections A and C).
   2. Calculate complex I to IV (CI-CIV) activity by subtracting residual activity from the basal values (measurement points 1 and 2).
   3. Subtract residual activity from the values post injection of the CII substrate succinate (injection B) to obtain CII-CIII-CIV activity.
   4. Calculate CIV activity by deducting the residual activity from the values obtained after injecting the cytochrome c-reducing agents, ascorbic acid and TMPD (injection D).
3. Represent all results using bar plots, as in Figure 4, to extract appropriate conclusions.

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 a...

Discussion

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 repro...

Materials

Name	Company	Catalog Number	Comments
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)