Key words

1 Introduction

Mitochondria have a central role in the ability of skeletal muscle to facilitate movement and regulate glucose homeostasis. Accordingly, mitochondrial dysfunction is involved in the pathogenesis of diseases such as diabetes [1, 2], sarcopenia [3–5], inclusion body myositis [6], and various mitochondrial myopathies [7–9]. Methods to accurately and reliably measure aerobic respiration can support studies examining how mitochondrial function is regulated in health and disease.

The Seahorse Bioscience XFe24 utilizes solid state sensor probes to measure the concentration of oxygen surrounding live cells. The rate of oxygen utilization by the cells in indicative of aerobic respiration . This system is integrated into a microplate format for analyzing multiple samples simultaneously. Its most well established use has been the analysis of tissue culture cells [10]. Following baseline measurements, cells can then be treated with drugs or substrates to further dissect specific aspects of their metabolic phenotype [11].

Measuring respiration from tissues, while advantageous for studying animal disease models, has been more technically challenging. Isolating mitochondria from tissues is the most straightforward way to do this, but they can undergo damage during the isolation process and differential centrifugation can bias the analyzed population [12]. In addition, mitochondrial function has been linked to interactions with the intracellular environment [13–15]. Alternatively, researchers have used permeabilized skeletal muscle single fibers or permeabilized tissues. Both of these strategies keep intracellular structures intact and enable the use of a myriad of substrates, inhibitors, and stimulants to dissect mitochondrial function [16, 17]. However, tissue permeabilization, while allowing compounds into cells, also releases the cytoplasm and any pertinent cytosolic factors from cells. This strategy can yield vast amounts of data from muscle tissue, as long as cytoplasmic components are not pertinent to the conclusions being drawn.

Here, we present a methodology for measuring the oxygen consumption rate (OCR) of intact, non-permeabilized skeletal muscle tissue using the Seahorse Bioscience XFe24. The protocol we describe here does not offer the range of data that could be obtained from drug and substrate treatments, though our preliminary data suggest that treatment with the uncoupling agent carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) is a feasible and useful tool for analyzing muscle tissue respiration. This protocol provides a measure of basal respiration from muscle tissues that retain more of their relevant physiological traits. We have, as an example, compared soleus and extensor digitorum longus (EDL) muscles and observed an expectedly higher OCR in the soleus (Fig. 1), where there are proportionally more slow fibers and mitochondria . In addition, this methodology can be applied to muscles of disease models and muscles electroporated with gene expression plasmids. As mentioned above, aging has been associated with a decline in mitochondrial function. This decline has been measured experimentally through enzymatic staining of muscle cross sections [18], gene expression profiling [19], and vO2-max measurements [5]. Here, we have compared tibialis anterior (TA) muscles of 5-month and 24-month-old mice and found a significant reduction in OCR associated with aging (Fig. 2). In summary, this methodology can discern differences in the basal respiration rate of healthy, diseased, and genetically modified intact skeletal muscle tissues.

Fig. 1
figure 1

OCR measurements reflect differences between slow and fast muscles. OCR was measured from soleus and EDL muscles of 8-week-old mice (see Note 5 )

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Fig. 2
figure 2

Aging reduces skeletal muscle OCR. The OCR from TA muscles of 5-month and 24-month-old mice were measured and normalized to dry tissue weight

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2 Materials

2.1 Oxygen Consumption Rate Measurements

  1. 1.

    XFe24 Extracellular Flux Analyzer (Seahorse Bioscience).

  2. 2.

    XFe24 Assay Kit sensor plate.

  3. 3.

    XF Calibrant Solution.

  4. 4.

    XF24 Islet Capture Microplate with capture screens.

  5. 5.

    XF Islet Capture Screen Insert Tool.

2.2 Tissue Preparation

  1. 1.

    Dissection instruments: scissors, forceps, needles, tray.

  2. 2.

    70 % ethanol.

  3. 3.

    Razor blade.

  4. 4.

    60 mm petri dishes.

  5. 5.

    Complete Assay Medium: XF Assay Medium, 2 mM l-glutamine, 1 mM sodium pyruvate, 10 mM glucose. For working solution, combine 98 mL of XF Assay Medium, 1 mL of 200 mM l-glutamine, 1 mL of 100 mM sodium pyruvate, and 0.180 g of glucose. Adjust pH to 7.4 at 37 °C with sodium hydroxide (see Notes 1 – 3 ).

2.3 Normalization

  1. 1.

    60 °C oven.

  2. 2.

    Analytical balance.

3 Methods

3.1 XFe24 Assay Kit Sensor Plate Preparation

  1. 1.

    One day before the experiment, prepare an extracellular flux assay sensor plate by adding 1 mL of calibrant solution to the bottom of each well, placing the sensors into the wells, and storing overnight in a 37 °C non-CO2 incubator. Keep the spacer between the sensors and plate until the calibration (Subheading 3.6, step 2).

  2. 2.

    Check that the XFe24 Analyzer is on and set to 37 °C.

3.2 Tissue Preparation

  1. 1.

    Aliquot 100 μL of Complete Assay Medium into each well of an Islet Capture Microplate and place the plate in the 37 °C non-CO2 incubator.

  2. 2.

    Prepare a 60 mm petri dish for each tissue sample by adding 10 mL of Complete Assay Medium and labeling. Place petri dishes in the 37 °C non-CO2 incubator.

  3. 3.

    Prepare one additional 60 mm petri dish with 10 mL Complete Assay Medium and the islet capture screens and place in the 37 °C non-CO2 incubator.

  4. 4.

    Place remaining Complete Assay Medium in the 37 °C non-CO2 incubator.

  5. 5.

    Dissect muscles and place each in its own 60 mm petri dish containing warm Complete Assay Medium. Keep the dishes at 37 °C while dissecting the other muscle samples.

  6. 6.

    For each muscle, remove tendons with a razor, then cut muscle sections measuring approximately 2 × 2 × 1 mm. As you cut each piece, place it back in the warm medium (see Notes 4 and 5 ).

3.3 Assay Plate Setup

  1. 1.

    Add one tissue piece per microplate well, keeping indicated wells empty for background measurements (Fig. 3).

    Fig. 3
    figure 3

    Assay plate loading scheme. Wells labeled as “background” should contain medium and a screen, but no tissue. This will serve as a control for temperature fluctuations throughout an experiment and across the plate

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  2. 2.

    Slide a screen into each well using forceps, being careful that the tissue is centered under the screen and there is no trapped air underneath (see Note 6 ). Screens should also be added to background wells.

  3. 3.

    Press down on the screen with the XF Islet Capture Screen Insert Tool so that it is firmly seeded at the bottom of the well.

  4. 4.

    Add 600 μL of Complete Assay Medium to each well.

  5. 5.

    Place in the 37 °C non-CO2 incubator for 1 h.

3.4 XFe24 Template Setup

  1. 1.

    Design a new blank template.

  2. 2.

    Under the Group Definitions tab, click on the Assay Conditions screen. Define a separate “cell type” for each sample.

  3. 3.

    Create a Well Group for each “cell type.” Pair a “cell type” to each Well Group.

  4. 4.

    On the Plate Map screen, assign each Well Group to its corresponding wells.

  5. 5.

    Under the Instrument Protocol tab, edit the measurement details. Set measurement procedure to MIX 3 min, WAIT 5 min, and READ 2 min. Repeat basal measurement at least four times (see Note 7 ).

  6. 6.

    Under the Review and Run tab, be sure the Calibrate and Equilibrate boxes are checked.

3.5 XF e24 Measurements

  1. 1.

    Click on Start Run.

  2. 2.

    Start the protocol by calibrating the sensors. Remove the spacer and place the sensors back in the wells containing calibration buffer. When prompted, load the sensors with wells onto the tray that slides out of the Analyzer.

  3. 3.

    When the 1 h tissue incubation is complete, prompt the Analyzer to proceed and replace the calibration buffer plate with the tissue samples.

3.6 Normalization

  1. 1.

    After measurements are completed, remove screens and transfer the tissues to labeled Eppendorf tubes.

  2. 2.

    Incubate uncapped tubes in a 60 °C oven for 48 h.

  3. 3.

    Weigh dried tissues.

  4. 4.

    Calculate OCR per mg of dry tissue for each sample.

4 Notes

  1. 1.

    Adjusting the pH of the Complete Assay Medium should be done at 37 °C and in equilibrium with ambient air, so that the adjusted pH will remain constant during the experiment. A glass beaker or conical tube stabilized in a 37 °C water bath works for this.

  2. 2.

    Seahorse Assay Medium does not contain any buffering agent like bicarbonate, so the pH should be adjusted in small increments.

  3. 3.

    Prepared Complete Assay Medium can be filter-sterilized and stored up to 1 week at 4 °C without having to check the pH again. For longer storage, check the pH before using.

  4. 4.

    Skeletal muscle fiber types are not evenly distributed. In general, deep fibers tend to be more oxidative than more superficial fibers. Therefore, care should be taken to take similar representative samples when comparing tissue samples, by comparing either total cross sections or similar anatomical regions. We have found it useful to analyze relatively small muscles such as the tibialis anterior or soleus when this is possible and appropriate for the experimental design.

  5. 5.

    When developing this protocol, one concern was whether slicing through fibers would disturb OCR measurements. When comparing fully intact EDL muscles versus EDL muscles cut in half, measurements normalized to dry tissue weight were nearly identical.

  6. 6.

    The screens are “U” or cup shaped and should be placed into the wells with the “U” open end facing up. If it is hard to determine which way they are facing, the screens will be substantially easier to pick up with the forceps if they are oriented the right way.

  7. 7.

    Repeated measurements should be fairly consistent. If they are progressively declining, you might try increasing the MIX and WAIT times. In addition, increase the number of repeated measures to see whether the readings stabilize after the tissues equilibrate with the medium. We have found that incubating the plate and tissues less than an hour prior to measurements gives declining OCR values over time, until they eventually stabilize. These early values are unreliable, so care should be taken to allow tissues to equilibrate. We have observed stable OCR values up to 3 hours after dissection.