Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Summary

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Abstract

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Introduction

Mitochondria are organelles, approximately the size of bacteria, found in eukaryotic cells. They are unique organelles because they contain DNA and have two membranes, an outer and inner one. Mitochondria's outer and inner membranes are separated by an intermembrane space, and the inner membrane folds into structures called cristae around the innermost compartment, called the matrix. These cristae increase the inner membrane's surface area so that multiple processes that use the cristae can occur simultaneously. While mitochondria are involved in many cellular functions such as controlling apoptosis and housing multiple metabolic pathways, their vital role in the production of ATP is essential for cell survival. In fact, 90% of a cell's energy is derived from mitochondria¹. ATP production involves the generation of an electrochemical difference across the outer and inner membranes, termed the mitochondrial membrane potential (Δψ), which arises as H⁺ ions are pumped from the matrix into the intermembrane space. ATP production is ultimately harnessed during the oxidation of reducing equivalents via electron movement through the mitochondrial respiratory chain (ETC). The final electron acceptor is molecular oxygen (O₂). As oxygen is consumed, the H⁺ concentration differential builds up to its maximum, at which point H⁺ ions move down their concentration gradient from the intermembrane space to the matrix by passing through the ATP synthase complex. The movement of H⁺ ions causes a conformational change in ATP synthase, and ADP is brought into proximity with inorganic phosphate to react and generate ATP. Finally, ATP is translocated out of the mitochondrial matrix into the cytosol and can either be stored or used to facilitate reactions due to the large amount of free energy released during the hydrolysis of its phosphates. This whole process is termed oxidative phosphorylation, and since oxygen is consumed, mitochondria are said to respire².

The buildup and strength of Δψ, the amount of O₂ reduced (termed oxygen consumption), as well as the generation of ATP can all be used as indications of cell health. Mitochondrial functional studies, such as measurement of Δψ, total ATP content and production, and oxygen consumption can be quantified either by traditional biochemical methods or fluorescence and luminescence in plate-based assays. For example, mitochondrial membrane potential can be compared among different samples using fluorescent dyes such as tetramethylrhodamine ethyl ester, which binds specifically to mitochondria. ATP generation can be monitored by adding a luminescent protein to a reaction whose changes correlate to ATP concentration. Quantification of oxygen consumption rates, or absolute rates of respiration, during OXPHOS, can help elucidate the causes of disparities in mitochondrial function and energy metabolism. Oxygen consumption assessment can be used to calculate respiratory control ratios (RCRs). RCR values describe the ability of mitochondria to make ATP in response to the influx of ADP, which is the main function of mitochondria. RCR values signify the overall condition of isolated mitochondria and allow for the comparison of responses to different experimental treatments. Differences in RCR values may represent mitochondrial dysfunction or indicate a biological difference between different mitochondria isolated from two or more sources. Another important measure of function in isolated mitochondria is mitochondrial efficiency defined as moles of ATP synthesized per moles of O₂, or the P/O ratio³.

Given the amount of information that can be gathered from measuring mitochondrial parameters and various instances in which this information can be utilized, the ability to efficiently gather functional data can be useful in many different research areas. Mitochondrial oxygen consumption measurements have been performed for decades with very specific instrumentation—using a Clark electrode, which can be limited by the sample size necessary to carry out measurements, and more recently, sophisticated instruments that can measure mitochondrial respiration and multiple other parameters but can be cost-prohibitive. This protocol is an adapted alternative approach using an oxygen-sensitive phosphorescent probe (MitoXpress)^4,5. The probe signal is detected with a plate reader in time-resolved fluorescence mode for continuous measurements over time. Phosphorescence has a larger energy difference between the absorbed and emitted photon compared to fluorescence and therefore, is better suited for continuously monitoring changes in signal. This enables almost any lab to perform these measurements, not just those that focus on mitochondrial metabolism or who can afford highly specialized equipment. The model system we utilize is isolated mitochondria from three tree lizards, two parental species and one introgressed (containing nuclear DNA from one parental species and mitochondria from the other—hybrids). These lizards were chosen because we hypothesized there are metabolic and energy consequences for hybrids having different nuclear and mitochondrial DNA sources. We utilized a commercially available assay kit with a multi-mode plate reader that can increase access to this type of assay to more researchers and research fields.

Protocol

Lizards were euthanized by CO₂ asphyxiation followed by immediate decapitation in accordance with policies outlined by The Office of Animal Laboratory Welfare and Elon's Institutional Animal Care and Use Committee guidelines.

1. Isolation of mitochondria⁶

NOTE: Keep all solutions cold (Table 1) and samples on ice throughout these steps.

1. Remove the liver, weigh it, and then rinse it with ~3 mL of ice-cold phosphate-buffered saline (1x, -/-).
2. Mince the liver in 1 mL of L-MIB with a fresh razor blade.
3. Bring the total volume of tissue plus L-MIB to 2 mL and mechanically disrupt the liver cells for four passes with a Dounce homogenizer.
4. Spin the homogenate at 300 × g (37 °C, 10 min).
5. Transfer the supernatant into a fresh tube and place it on ice.
6. Resuspend the pellet in 2 mL of L-MIB, rehomogenize, and spin again at 300 × g (37 °C, 10 minutes).
7. Combine the supernatants from both spins and centrifuge at 10,000 × g (37 °C, 10 min).
8. Resuspend the pellet in ~0.350 mL of L-MIB.
9. Determine the amount of total protein (mg of mitochondria/mL of L-MIB) to use as an approximation for mitochondrial content^4,5,6.

2. Oxygen consumption

1. Prewarm all the solutions used (Table 1) in the following steps to 30 °C in a water bath.
2. Dilute the mitochondria in LEB to 6 mg/mL based on the results of the protein concentration assay in step 1.9.
3. Set up the assay in a sterile 96-well black-wall, clear bottom plate (Table 2).
   1. Add 50 µL of the sample (L-EB buffer or mitochondria sample) to the appropriate wells.
   2. Add 50 µL of treatment (L-EB, Glutamate/Malate, or Glutamate/Malate w/ADP).
   3. Dilute the probe stock 1:10 in L-EB and then add 100 µL of the fluorescent probe to every well.
   4. Gently add 50 µL of heavy mineral oil to every well to exclude ambient oxygen.
4. Read fluorescent measurements from the bottom of the plate at 380/650 nm excitation/emission every 1.5 min for 45 min. Use kinetic mode with a time delay of 30,000 µs and a measurement window of 100 µs.

3. Data analysis

1. Export the raw data file from the plate reader computer as a .xls file.
2. Open the file, then copy and paste the raw data into a new tab, and label the rows and columns appropriately.
3. Plot the L-EB buffer-only to the control sample and L-EB + G/M as relative fluorescent units (RFU) versus time.
   NOTE: These lines should be relatively flat as there are no mitochondria present, and therefore, no change in oxygen concentration should be detected.
4. Plot the L-EB + G/M sample values as RFU versus time.
5. Determine where the readings become more consistent and flatten out for the controls.
   NOTE: This is where data analysis for experimental samples should commence (marked by arrows in Figure 1A). Reactions can take 10-15 min to flatten out and reach their maximum.
6. Make a new plot only containing the data after the time point established in step 3.5 (called "trimmed" data) and add the best-fit line to visualize the raw data to be analyzed.
7. Calculate the average RFU value of the trimmed control buffer-only data (highlighted in yellow). This value is used as the minimum RFU value as the baseline in the oxygen calculation below.
8. Calculation of oxygen consumption rate (µM O₂/min)
   1. Copy and paste the linear region of the raw data from step 3.6 (i.e., the trimmed data) to determine the concentration of oxygen in the mitochondrial samples in G/M + ADP treatment at each time point^7,8,9,10.
   2. Use equation (1)⁵ to determine the oxygen concentration at each time point. [O₂]_a is the oxygen concentration in air-saturated buffer (235 mM at 30 °C). I(t), Ia, and Io are the fluorescent signal of the sample + probe at time t (e.g., sample + G/M + ADP), the average signal of the probe in air-saturated buffer calculated in step 3.7, and the maximal signal in deoxygenated buffer (set at the maximum signal achievable), respectively.
           (1)
   3. Plot the [O₂]_t values calculated in step 3.8.2 versus time and add a linear trendline along with the equation of the best-fit line.
      NOTE: Use the slope of each line as the oxygen consumption rate. Duplicates or triplicates for any sample can be aggregated and used for the comparison of the rate of consumption.
9. Calculate the RCR by dividing mitochondrial respiration with and without ADP, which represent states 3 and 2, respectively.

Results

Oxygen consumption rate and mitochondrial RCR were determined from the mitochondria of three different lizards using an assay kit with a phosphorescent oxygen-sensing probe and a standard fluorescence plate reader. Previous research established that the probe in this kit directly correlates to oxygen consumption, where phosphorescence is quenched by molecular oxygen and the fluorescent signal increases as oxygen levels decrease due to mitochondrial respiration^7,11

Discussion

Measuring mitochondrial function is useful when comparing different samples, such as disease versus non-disease states, different tissue types from the same animal, or between different sample types. We used the later comparison to test our hypothesis that there is a metabolic consequence to hybrid tree lizards that have introgressed mitochondria. There are a variety of ways to ascertain mitochondria function experimentally, including quantification of Δψ, total ATP content, ATP production, and respiratory cont...

Materials

Name	Company	Catalog Number	Comments
96-well black/optical bottom plates	Thermo Fisher	265301	Untreated black-wall plates with clear bottoms.
ADP	Sigma	A2754	Dilute 100 µM stock with EB immediately before use.
BSA	Thermo Fisher	BP1600-100	Make 2 mg/mL stock in water for protein assay.
Dulbeccos 1x PBS (-/-)	Sigma	D8537	Make sure the PBS is without Mg2+ or Ca2+ ions.
EGTA	Sigma	E3889
K2HPO4	Sigma	P3786
KH2PO4	Sigma	P0662
L-glutamic acid	Sigma	G1251
L-glutamic acid potassium salt	Sigma	S372226
L-malic acid	Sigma	M8304
L-malic acid mono-potassium salt	Sigma	49601
MitoXpress oxygen consumption kit	Agilent	MX-200-4	Kit contains probe stock and HS mineral oil.
MOPS	Sigma	M3183
Protien Assay Dye (5x)	BioRad	500-0006	Any protein assay can substitute.
R version 3.3	R Core Development Team 2016
Thermomax microplate reader EnSpire Multi-mode Plate reader and software	PerkinElmer		Standard fluorescent plate-reader
Trisma base	Sigma	T6066	Any version of Tris base can be utilized.