Analyzing Mitochondrial Function in a <em>Drosophila melanogaster</em> PINK1<sup>B9</sup>-Null Mutant Using High-resolution Respirometry

Paula Michelotti; T&#226;mie Duarte; Cristiane L. Dalla Corte

doi:10.3791/65664

JoVE Journal > Biochemistry

Biochemistry

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1^B9-Null Mutant Using High-resolution Respirometry

Published: November 10, 2023

doi:

10.3791/65664

Paula Michelotti*^1,2, Tâmie Duarte*^1,2, Cristiane L. Dalla Corte^1,2

¹Laboratory of Experimental Biochemistry and Toxicology, Department of Biochemistry and Molecular Biology, Center of Natural and Exact Sciences,Federal University of Santa Maria, ²Graduate Program in Biological Sciences: Toxicological Biochemistry,Federal University of Santa Maria

Summary

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1^B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Abstract

Neurodegenerative diseases, including Parkinson's Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.

This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1^B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Introduction

Mitochondria are cellular organelles that control important functions, including apoptotic regulation, calcium homeostasis, and participation in biosynthetic pathways. By possessing autonomous genetic material, they are capable of contributing to cellular maintenance and repair processes. Their structure houses the electron transport chain and oxidative phosphorylation, both crucial for cellular energy¹^,²^,³. In particular, energy control is achieved through adenosine triphosphate (ATP) production via oxidative phosphorylation (OXPHOS)². Disruption of energy metabolism with impairment of mitochondrial functions occurs both in cell survival and death⁴^,⁵, frequently associated with a wide range of human pathologies, such as cancer, and neurodegenerative diseases such as Parkinson's Disease (PD)³^,⁶.

PD is a chronic, progressive, and neurological disorder. The primary cause of this disease is the death of brain cells, especially in the substantia nigra, which are responsible for the production of the neurotransmitter dopamine, which controls movement⁶^,⁷^,⁸. The earliest observation that linked Parkinsonism to mitochondrial dysfunction was made in 1988, in experimental models using toxins that inhibit the respiratory chain Complex I⁹.

Currently, there are several methods to evaluate mitochondrial dysfunction¹⁰^,¹¹^,¹²^,¹³; however, compared to conventional approaches, high-resolution respirometry (HRR) presents superior sensitivity and advantages¹³^,¹⁴. For example, HRR protocols allow the evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes¹⁴^,¹⁵. Mitochondrial dysfunctions can be assessed in intact cells, isolated mitochondria, or even ex vivo¹⁰^,¹¹^,¹³^,¹⁴.

Mitochondrial dysfunctions are closely associated with many pathological and physiological processes. It is therefore important to study mitochondrial physiology and bioenergetics using genetically and experimentally tractable model systems. In this regard, research on Drosophila melanogaster, the fruit fly, has several advantages. This model shares fundamental cellular characteristics and processes with humans, including the use of DNA as genetic material, common organelles, and conserved molecular pathways involved in development, immunity, and cell signaling. In addition, fruit flies have a rapid life cycle, easy maintenance, low cost, high throughput, and fewer ethical concerns, thus constituting an invaluable tool for dissecting complex cellular processes¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁰.

Furthermore, a homolog of the PTEN-induced putative kinase 1 (pink1) gene is expressed in D. melanogaster. It plays a crucial role in the removal of damaged mitochondria through the process of mitophagy⁸. In humans, mutations in this gene predispose individuals to an autosomal recessive familial form of PD associated with mitochondrial dysfunction⁸^,²¹^,²²^,²³. Consequently, the fruit fly is a powerful animal model for studies on the pathophysiology of PD and screening of drug candidates focusing on mitochondrial dysfunction and bioenergetics. Therefore, the present work explains how to analyze mitochondrial function in a model of PD from D. melanogaster using the HRR technique in the OROBOROS with the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Protocol

We used the strains w¹¹¹⁸ (white) and w[*] Pink1[B9]/FM7i, P{w[+mC]=ActGFP}JMR3 (referred to as Pink1^B9) (FlyBase ID: FBgn0029891) from the Bloomington Drosophila stock center (ID number: 34749). In this study, male D. melanogaster PINK1^B9-null mutants are compared with male D. melanogaster from the w¹¹¹⁸ strain, which is used as a control group (genetic background). Other parameters must be analyzed concomitantly with the respirometry experiments to ensure that the flies have the correct genotype (Pink1^B9/Y), such as thorax deformities and locomotion problems, which are well described for pink1^B9 mutant flies²⁴^,²⁵^,²⁶.

1. Animals and housing

Keep the flies in glass bottles containing 10 mL of standard diet (yeast 1.73%, soy flour 0.9%, corn flour 7.3%, agar 0.5%, corn syrup 7.6%, and propionic acid 0.48%) at constant temperature and humidity (25 °C and 60%, respectively), with a 12 h:12 h light/dark cycle.
For each experiment, use two flies aged 1-3 days post-eclosion.
NOTE: The PINK1^B9-null mutant virgin female flies were crossed with w¹¹¹⁸ males to obtain the F1 males with genotype Pink1^B9/Y.

2. Sample preparation

Prepare the MiR05 buffer as described in Table 1.
Anesthetize the flies on ice and transfer them to microcentrifuge tubes (two flies per tube).
In each tube, add 200 µL of chilled MiR05 buffer and homogenize the flies. Homogenize manually, applying gentle pressure (approximately 4-6 strokes of the pestle) to prevent the disintegration of mitochondria.

3. High-resolution respirometry calibration of polarographic oxygen sensors

NOTE: The OROBOROS chambers have a total volume of approximately 2 mL. Calibration is required to ensure the oxygen flux is close to 0 pmol to start the assay.

Starting the calibration, add 2 mL of MiR05 to the chamber. Cover the chamber with the stopper leaving no air bubbles; then, carefully pull the stopper to form one single air bubble.
Open the OROBOROS Dat.Lab software. Enter a temperature of 25 °C in the Block temperature field and click on Connect to Oxygraph-2k (Figure 1A).
Click and select the folder where the calibration is to be saved and click on Save.
When a new window opens, name the experiment and the samples (if calibrating, simply enter the name calibration) and click on Save.
Go to the Layout menu and choose the first option: 01 Calibration Exp. Gr.3 – Temp as shown in Figure 1B.
Wait for the program to redirect to the calibration layout, wait until the red line shows a standard straight line with points around 0 pmol. Then, select two points: one for chamber A and the other for chamber B (Figure 1C) when the red line is exactly at zero.
NOTE: The selected points must have oxygen flux (red line) around 0.
1. To mark the points of both chambers, select O₂ concentration on the right of the screen, select the marked point, and copy both the temperature and barometric pressure values relative to the point. Paste these values in the boxes below and click on Calibrate and Copy to Clipboard in the lower right corner of the screen as shown in Figure 2.
2. Carry out this process for both chambers (A and B), then go to the File menu and select Save and Disconnect.
  NOTE: After the calibration process, the program will redirect the user to the home screen so that the software will be ready to start the HRR test. For this test, we will use the SUIT protocol.

4. SUIT protocol

With the calibration completed, remove the stoppers and open the chambers.
Wash the stoppers (holding the tip that does not touch the sample) and chambers with 100% ethanol, 70% ethanol, and distilled water, in that order.
NOTE: Repeat the process for every sample change.
Homogenize the flies in 200 µL of buffer, place all sample content in the chamber using a micropipette, and relocate the stopper in the chamber, taking care to not create air bubbles.
NOTE: If air bubbles form, remove the stopper gently and add 100 µL of MIR05 buffer.
Click on the Layout menu and select the option 05 Flow by Corrected Volume as shown in Figure 1B.
Wait to be redirected to a new window. Click on Save to select the folder where the experiment is to be saved.
Wait for another new window to open. Name the experiment in the space Experimental Code. Then, name each sample in its respective chamber A and B in the Sample field, and set the field Unit to unit.
NOTE: Other units can also be chosen, for example, Millions of cells or mg.
Considering that this protocol uses two flies (2 units) and the chamber volume is 2 mL, in the Concentration field, define 1 per mL (Figure 1D).
NOTE: Here, the sample amount was normalized by the number of flies; however, this normalization may be performed by the protein content of the sample, mitochondrial DNA quantification, or activity of citrate synthase.
When the oxygen flux signal (red line) is steady on the positive value, start the HRR protocol with the titration of substrates, inhibitors, and uncouplers (Figure 3). All the subsequent reagents and equipment are shown in Table 1.
1. Add digitonin (5 μg/mL) to permeabilize the mitochondrial membrane.
2. Add pyruvate (5 mM), malate (2 mM), and proline (10 mM) substrates and wait until the oxygen flux increases and stabilizes. To mark the event for each reagent addition, press the F4 key on the keyboard and enter the name of each reagent.
3. Add ADP (5mM) to couple the mitochondrial respiratory chain and wait for the increase and stabilization of the oxygen flux.
4. Add succinate (10 mM) and wait for the increase and stabilization of the oxygen flux.
5. Add Oligomycin (2.5 µM) to inhibit ATP synthase and look for a decrease in oxygen flux.
6. Wait until the red line stabilizes and uncouple mitochondrial electron transfer using Carbonyl-4-(trifluoromethoxy) phenylhydrazone cyanide (FCCP) with titers of 0.25 µM until the maximum oxygen consumption is reached, demonstrated by the rise of the red line. After stabilization of the oxygen flux, start adding the inhibitors of each complex, one at a time.
7. Add Rotenone (0.5 µM), a complex I inhibitor. Wait for the decrease and stabilization of the oxygen flux to add the next inhibitor.
8. Add Malonate (5 mM), a complex II inhibitor. Wait for the decrease and stabilization of the oxygen flux to add the next inhibitor.
9. Finally, add Antimycin (2.5 µM), a complex III inhibitor. Wait for the decrease followed by the increase and stabilization of the oxygen flux.
  NOTE: The oxygen flux should stabilize at a positive value but below the value for the last inhibition of mitochondrial protein complexes.
At the end of the analysis, remove all the content of the chambers using a micropipette. Store at -20 °C for future analysis, when necessary.

5. Data analysis

Utilize an appropriate statistical software package for data analysis.
Perform t-tests to assess differences between the two groups. For situations involving multiple treatments, use Analysis of Variance (ANOVA) as a statistical test ²⁷.
Extract the O₂ flux values from the graph generated by the software. OXPHOS CI refers to the value of O₂ flux after adding ADP. OXPHOS CII is obtained by subtracting OXPHOS CI&CII – OXPHOS CI. OXPHOS CI&CII is the value of O₂ flux extracted after the addition of succinate. ETS CI&CII is the O₂ flux value after adding FCCP. ETS CI = FCCP – Rotenone and ETS CII = Rotenone – Malonate.
Calculate ATP synthesis as the difference between the OXPHOS (P) and LEAK (L) (P – L)²⁸.
Determine the Respiratory Control Ratio (RCR) by calculating the ratio OXPHOS/LEAK, where RCR = P/L.¹¹

Representative Results

Here, we that O₂ flux in OXPHOS CI (P = 0.0341) and OXPHOS CI&II (P = 0.0392) states is reduced in PINK1^B9 null flies when compared to control flies (Figure 4). This result was also observed in previous findings from our group²⁹^,³⁰.

CI and CII are key components of the electron transport system (ETS), in which CI is responsible for the transfer of electrons from NADH to ubiquinone, while CII transfers electrons from succinate to ubiquinone³¹^,³²^,³³. PINK1^B9 null flies showed lower O₂ flux in ETS CI (P = 0.0338), ETS CII (P = 0.0457), and ETS CI&II (P = 0.0247) states (Figure 5). These results indicate that the electron transfer system is impaired in flies lacking the pink1 gene and the O₂ flux in both OXPHOS and ETS stages are dependent on CI and CI&CII, consistent with other works demonstrating reduced CI activity in pink1^-/- models³³^,³⁴^,³⁵.

Furthermore, the proton gradient across the mitochondrial inner membrane is essential for the synthesis of ATP²⁸. The decrease in O₂ flux in ETS CI and ETS CII indicates a disruption in the flux of electrons along the ETS. This disruption in the flux of electrons affects the OXPHOS process leading to reduced ATP synthesis. There was also a significant decrease in O₂ consumption related to ATP synthesis (P = 0.0280) in PINK1^B9 null flies when compared to control flies (Figure 6B). A decrease in ATP synthesis in D. melanogaster can have significant effects on energy metabolism, cellular processes, and overall physiological functions. In addition, the efficiency of the OXPHOS process can be quantified by an index known as RCR, which reflects the tightness of the coupling between respiration and phosphorylation. Therefore, RCR reduction (P = 0.0432) indicates mitochondrial uncoupling, which may affect the OXPHOS process suggesting that the mitochondria are less efficient at utilizing oxygen and producing ATP (Figure 6C). These results may impact the fruit fly's growth, development, locomotion, reproduction, and overall health and contribute to the pathogenesis of certain neurodegenerative diseases, including PD⁸^,²⁸^,²⁹^,³².

Figure 1: Layouts in OROBOROS Dat.Lab software. Please click here to view a larger version of this figure.

Figure 2: Steps of calibration of chambers in OROBOROS Dat.Lab software. Please click here to view a larger version of this figure.

Figure 3: SUIT protocol demonstrating the main points of substrate and inhibitor addition. Firstly, digitonin (DIG) is added followed by complex I specific substrates: malate, pyruvate, and proline (MPP), ATP synthase substrate (ADP), and then, substrate for complex II: succinate (S). Subsequently, the ATP synthase inhibitor: oligomycin (OMY) is added, followed by the uncoupler Carbonyl-4-(trifluoromethoxy) phenylhydrazone cyanide (F), and complex I, II, and III inhibitors: rotenone (R), malonate (MNA), and antimycin (AMA)³⁶. Please click here to view a larger version of this figure.

Figure 4: High-resolution respirometry comparing w¹¹¹⁸ and PINK1^B9 flies. (A) OXPHOS CI, (B) OXPHOS CII, and (C) OXPHOS CI&CII. Data are presented as mean ± S.E.M. and analyzed using t-test. n = 5-9. p < 0.05. Abbreviations: OXPHOS = oxidative phosphorylation; CI = complex I; CII = complex II. Please click here to view a larger version of this figure.

Figure 5: High-resolution respirometry comparing w¹¹¹⁸ and PINK1^B9flies. (A) ETS CI, (B) ETS CII, and (C) ETS CI&CII. Data are presented as mean ± S.E.M. and analyzed by t-test. n = 5-9. p < 0.05. Abbreviations: ETS = electron transport system; CI = complex I; CII = complex II. Please click here to view a larger version of this figure.

Figure 6: High-resolution respirometry comparing w¹¹¹⁸ and PINK1^B9 flies. (A) LEAK, (B) ATP synthesis, and (C) respiratory control ratio. Data are presented as mean ± S.E.M. and analyzed by t-test. n = 5-9. p < 0.05. Abbreviation: RCR = respiratory control ratio. Please click here to view a larger version of this figure.

Discussion

HRR is a powerful technique for studying mitochondrial respiration and energy metabolism in D. melanogaster and other organisms. It provides a detailed and quantitative assessment of mitochondrial function, allowing researchers to gain insights into the bioenergetics of the cells. The protocol presented here describes the evaluation of mitochondrial respiratory chain function and the activity of specific mitochondrial complexes using the SUIT protocol in D. melanogaster. The SUIT protocol involves systematically manipulating various substrates, uncouplers, and inhibitors to examine different aspects of mitochondrial respiration.

The technique described allows the assessment of respiratory inhibition resulting from effects on the OXPHOS or the ETS, dehydrogenase activity (CI and CII), and membrane integrity (coupling of OXPHOS). Here, we performed the experiments using PINK1^B9-null flies to study mitochondrial dysfunction since it is associated with PD³⁴^,³⁵. However, this protocol can be useful for different disease models, drug treatments, and toxicology studies.

In addition, the sample preparation may be adapted to fit experimental requirements³⁶. A critical step in the respirometry protocol is sample preparation. It is critical that the correct protocol be established for the type of sample (cell, isolated mitochondria, homogenate), and it is important to consider the appropriate method for sample normalization (protein amount, DNA content, citrate synthase activity). It is also important to use the same buffer for sample preparation and the respirometry assay.

As the sample preparation protocol described here is simple, it does not usually pose problems for the technique. If another type of sample is chosen or its preparation changed, then careful standardization is necessary. Stirring and temperature stability affect the signal of the polarographic oxygen sensor, generating error in respiratory measurements using an oxygraph. Therefore, the correct calibration of the chamber constitutes an important step to reduce errors during the respiratory measurements.

This method for assessing mitochondrial function in fly samples offers several advantages over alternative approaches. One of the main strengths of HRR is its ability to provide direct and accurate measurements of oxygen consumption, allowing for a detailed analysis of mitochondrial function and cellular metabolism. Therefore, HRR is often used in research focused on understanding mitochondrial dysfunction, energy production, and cellular responses to different substrates or conditions. Furthermore, it is versatile – allowing the use of a wide variety of sample types, including isolated mitochondria – and requires small amounts of biological samples, which is useful when the sample amount is limited¹^,²^,³. Methods that use mitochondrial isolates, for instance, typically involve a substantial number of flies, ranging from 50 to 200 individuals, which makes the study with mutants difficult, as obtaining a large number of mutants for certain disease models may not be practical.

Similar to the HRR method, the Seahorse Bioscience Extracellular Flux Analyzer is a scientific tool used to measure oxygen consumption and extracellular acidification. However, they have different approaches and applications. The Seahorse Bioscience Extracellular Flux Analyzer quantifies instantaneous alterations in the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells. OCR is indicative of mitochondrial respiration and energy generation, whereas ECAR offers insights into glycolytic activity. Its principal function lies in the assessment of cellular metabolic dynamics under diverse physiological conditions, rendering it particularly instrumental in elucidating the intricate metabolic modulations associated with pathological contexts. In addition, Seahorse is designed for ease of use, allowing researchers to perform rapid metabolic assays³⁷^,³⁸^,³⁹. In contrast, the HRR requires specialized training and expertise to operate and analyze data effectively. It involves the use of an oxygen electrode to directly measure oxygen consumption by cells or mitochondria¹³^,¹⁴^,⁴⁰. An advantage of HRR is its versatility in designing different respirometry protocols, which is impracticable when using Seahorse. This versatility to design protocols associated with low cost makes the HRR method a viable choice for laboratories with limited funding. Another disadvantage of the respirometry system used in the present protocol is the impossibility of working with several samples simultaneously, which hampers the high-throughput analysis. Thus, experimental groups must be well-designed to allow the correct comparison between them. However, the advantages of this system lie in the fact that samples of any kind can be tested, from isolated cells to whole live organisms, such as C. elegans, for example⁴⁰.

In summary, HRR is a reliable method for studying mitochondrial function under physiological and pathological conditions⁴¹. Each experimental model has different characteristics and restrictions, requiring methodology and sample preparation adjustments to ensure reliable and meaningful data acquisition in mitochondrial respiration evaluation. This protocol offers researchers a reliable method to assess the effects of environmental factors, experimental interventions, or genetic mutations on mitochondrial function in D. melanogaster.

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors acknowledge the Brazilian agency Coordenação de Aperfeiçoamento de Pesquisa Pessoal de Nível Superior (CAPES EPIDEMIAS 09 #88887.505377/2020). P.M. (#88887.512821/2020-00) and T.D. (#88887.512883/2020-00) are research fellowship recipients.

Materials

ADP	Sigma-Aldrich	A5285	Adenosine 5′-diphosphate sodium sal (CAS number 72696-48-1); ≥95%; molecular weight = 501.31 g/mol.
Ágar	Kasv	K25-1800	For bacteriologal use
Antimycin-A	Sigma-Aldrich	A8674	Antimycin A from Streptomyces sp. (CAS number 1397-94-0); molecular weight 540 g/mol;
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A7030	Bovine Serum Albumin (CAS number 9048-46-8); pH 7,0 ≥ 98%
Datlab software	Oroboros Instruments, Innsbruck, Austria	20700	Software for data acquisition and analysis
Digitonin	Sigma-Aldrich	D 5628	CAS number 11024-24-1
Distilled water
Drosophila melanogaster strain w[*] Pink1[B9]/FM7i, P{w[+mC]=ActGFP}JMR3			Obtained from Bloomington Drosophila stock center
Drosophila melanogaster strain w¹¹¹⁸			Obtained from the Federal University of Santa Maria
EGTA	Sigma-Aldrich	E8145	Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (CAS number 13638-13-3); ≥97%; molecular weight =468.28 g/mol
FCCP	Sigma-Aldrich	C2920	Carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone (CAS number 370-86-5); ≥98% (TLC), powder
GraphPad Prism version 8.0.1.			Software for data acquisition and analysis
Hepes	Sigma-Aldrich	H4034	4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS number 7365-45-9); ≥99,5% (titration), cell cultured tested; molecular weight = 238.30 g/mol
High-resolution respirometer Oxygraph O2K	Oroboros Instruments, Innsbruck, Austria	10022-02	Startup O2K respirometer kit
KH₂PO4	Sigma-Aldrich	P5379	Monopotassium phosphate (CAS number 7778-77-0); Reagente Plus, molecular weigt = 136.09 g/mol
KOH	Sigma-Aldrich	211473	Potassium hydroxide (CAS number 1310-58-3); ACS reagent, ≥85%, pellets
Malate	Sigma-Aldrich	M1296	Malonic acid (CAS number 141-82-2); 99%, molecular weight = 104.06 g/mol). A solution is pH adjusted to approximately 7.0.
Malic acid	Sigma-Aldrich	M1000	(S)-(−)-2-Hydroxysuccinic acid (CAS number 97-67-6); ≥95% ; molecular weight = 134.09 g/mol
MES	Sigma-Aldrich	M3671	2-(N-Morpholino)ethanesulfonic acid (CAS number 4432-31-9); ≥99% (titration); molecular weight = 195.24 g/mol
MgCl₂	Sigma-Aldrich	M8266	Magnesium chloride (CAS number 7786-30-3); anhydrous, ≥98%, molecular weight = 95.21 g/mol
Microcentrifuge tubes	Eppendorf
O2K-Titration Set	Oroboros Instruments, Innsbruck, Austria	20820-03	Hamilton syringes with different volumes
Oligomycin	Sigma-Aldrich	O 4876	Oligomycin from Streptomyces diastatochromogenes (CAS number 1404-19-9); ≥90% total oligomycins basis (HPLC)
Pistil to homogenization
Proline	Sigma-Aldrich	P0380	L-Proline (CAS number 147-85-3); powder; 99%; molecular weight = 115.13 g/mol
Pyruvate	Sigma-Aldrich	P2256	Sodium pyruvate (CAS number 113-24-6), ≥99%; molecular weight = 110.04 g/mol
Rotenone	Sigma-Aldrich	R8875	Rotetone (CAS number 83-79-4); ≥95%, molecular weight 394.42 g/ mol
Succinate	Sigma-Aldrich	S 2378	Sodium succinate dibasic hexahydrate (CAS number 6106-21-4); ≥99%
Sucrose	Merck	107,651,000	Sucrose for microbiology use (CAS number 57-50-1)
Taurine	Sigma-Aldrich	T0625	CAS number 107-35-7

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Michelotti, P., Duarte, T., Dalla Corte, C. L. Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1^B9-Null Mutant Using High-resolution Respirometry. J. Vis. Exp. (201), e65664, doi:10.3791/65664 (2023).

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