Measurement of mitochondrial respiration in adherent cells by Seahorse XF96 Cell Mito Stress Test (2024)

Mitochondria play pivotal roles in cellular energy metabolism. Most of the intracellular adenosine triphosphate (ATP) is generated by mitochondrial respiration. The Cell Mito Stress Test is a common method to measure the key parameters of mitochondrial respiration. Here, we use the human cell line HK-2 as an example to present the procedures to quantify the oxygen consumption rate using a Seahorse XFe96 extracellular flux analyzer.

For complete details on the use and execution of this protocol, please refer to Ma etal. (2020).

• •

  Seahorse XF96 Cell Mito Stress Test protocol to measure mitochondrial respiration

• •

  Highlights the critical steps to be considered during the experiment

• •

  Lists the limitations and problems with the Seahorse XF96 Cell Mito Stress Test

1M NaOH (optional)

Transfection of HK-2 cells: day 1 and day 2

Timing: 2days

Cells are seeded in a sterile 6-well plate and transfect with SQSTM1/p62 overexpression plasmid and its control plasmid pcDNA3.1 (+).

• 1.

  Day 1: Plate 5× 10⁴ HK-2 cells in a sterile 6-well plate with 2mL of growth medium (DMEM medium supplemented with 10% fetal bovine serum) without antibiotics so that cells will be approximately 60%–80% confluency at the time of transfection.

• 2.

  Day 2: For each transfection samples, prepare complexes as follows:

  • a.

    Dilute 3.75μL Lipofectamine 3000 with 125μL Opti-MEM and mix gently.

  • b.

    Dilute 2500ng DNA in 125μL of Opti-MEM without serum, then add 5μL p3000 to it and mix gently.

  • c.

    Combine the diluted Lipofectamine 3000 with diluted DNA and mix gently. Incubate for 5min at 20°C–25°C. After 5min incubation, add the complexes to each well containing cells and medium. Incubate cells in a cell culture incubator with a humidified atmosphere of 5% CO₂ at 37°C for 24 h.

Note: For complete details of SQSTM1/p62 overexpression plasmid, please refer to our previous work (Ma etal., 2020). Compared with control plasmid, transfected with SQSTM1/p62 overexpression plasmid will increase the mitochondrial OXPHOS, which results in the increases of basal and maximal respiratory capacity, spare respiratory capacity, and ATP-linked OCR.

Note: It is not necessary to change the medium after transfection, but medium may be replaced after 24 h.

Seeding cells and hydrating sensory cartridge: day 3

Timing: 2–3 h

Seed the treated cells in the Seahorse XF96 Cell Culture Microplate, hydrate the XF extracellular flux sensory cartridge and turn on the Seahorse instrument (Figure1).

• 3.

  Cell seeding

  • a.

    Harvest and plate 6× 10³ HK-2 cells in 100μL growth medium (DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin) per well (Figure2A), except four background temperature correction wells (A1, A12, H1, and H12) (Figure3), which should be blanked with 100μL of growth medium.

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    Figure2

    Experimental operation in a well of a microplate and the interface connecting the Seahorse XFe96 extracellular flux analyzer to the computer

    (A) Cell seeding and liquid aspiration handling in a well of the Seahorse XF96 cell culture microplate.

    (B) In the lower-left corner of the Wave Controller software, you can verify the instrument connection status.

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    Figure3

    Four background correction wells in the Seahorse XF96 cell culture microplate (A1, A12, H1, H12)

    Be sure that background correction wells contain medium only (no cells).

  • b.

    Allow the plate to rest at 20°C–25°C in the tissue culture hood for 1 h. This can promote cell to distribute even and reduce edge effects for cells. Then, incubate the cells for 12–18h in a cell culture incubator.

Note: Optimal cell seeding density varies by cell type, but is typically between 5× 10³ and 4× 10⁴ cells per well for adherent cells. Generally, cell confluence between 80% and 90% will generate metabolic rates in the desirable range of the instrument.

Note: You must ensure your background wells do not contain cells.

Note: The key factors in cell seeding for accurate measurement are that the cells should be plated as uniform as possible and the plate must rest at 20°C–25°C in tissue culture hood for 1h to minimize the edge effect (Lundholt etal., 2003).

Note: Put the pipette tip on the edge of the lower well but not contact with the bottom of the well when seeding the cells. Do not resuspend the cells after seeding them in the XF96 Cell Culture Microplate.

• 4.

  Hydrate sensory cartridge

  • a.

    Separate the utility plate and sensory cartridge, and place the sensory cartridge upside down on the bench side to the utility plate (Figure4).

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    Figure4

    Cartridge lid, sensory cartridge, and utility plate

    Place sensory cartridge upside down on the bench and avoid contact with the sensors.

  • b.

    Fill each well of the utility plate with 200μL Seahorse XF Calibrant, then lower the sensory cartridge back onto the utility plate gently and avoid creating air bubbles.

  • c.

    Incubate the sensory cartridge in a non-CO₂, 37°C incubator for 12–18 h. Make sure the environment of the incubator is humidified.

Note: Be careful not to contact of the sensory cartridge and ensure submerging the sensors in the calibration.

• 5.

  Power on the Seahorse XFe96 Analyzer, non-CO₂, 37°C incubator and computer, then open the “Wave” software (Figure1) and click the “Heater on.”

Note: Observe whether the temperature in the software is rising to 37°C. When the icon in the lower-left corner of the “connected” turns green indicating that the instrument connection status is normal (Figure2B). The Seahorse instruments must turn on at least 5h prior to run the assay.

Day of the assay: day 4

Timing: 5–6 h

Prepare the Seahorse assay medium, cells, and XF Cell Stress Test Compounds for running the assay.

• 6.

  Prepare the Seahorse assay medium.

  • a.

    Turn on the water bath and warm to 37°C.

  • b.

    Add 1mL 100mM pyruvate solution, 1mL 200mM glutamine solution and 0.1g D-glucose in 98mL XF base medium. Filtrate the assay medium with 0.22μm filter.

    Reagent	Final concentration	Amount
    100mM pyruvate solution	1mM	1mL
    200mM glutamine solution	2mM	1mL
    D-Glucose	1 g/L	0.1 g
    XF base medium	–	98mL

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  • c.

    Warm the assay medium to 37°C and adjust to pH 7.4 with 1M NaOH.

    Note: The assay medium must be prepared when it will be used and keep 37°C prior to use. Do not prepare too much assay medium, 100mL is sufficient for one XF96 Cell Culture Microplate.

• 7.

  Wash cells

  • a.

    Retrieve the XF96 Cell Culture Microplate from the cell incubator.

  • b.

    Remove cell growth medium with 20μL remaining and replace with 180μL of assay medium, repeat 3 times.

  • c.

    The last time, aspirate 180μL of assay medium and replace with 160μL of assay medium.

  • d.

    To de-gas, incubate the XF96 Cell Culture Microplate in a 37°C incubator without CO₂ for 1 h.

Note: The cells must be incubated in a 37°C incubator without CO₂ for 1h to de-gas prior to replacing the XF96 Cell Culture Microplate on the tray.

• 8.

  Prepare and load XF Cell Stress Test Compounds

  • a.

    Suspend the compounds with assay medium according to Table 1.

    Table 1

    Stock solutions of oligomycin, FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) and rotenone/antimycin A

    Compound	Volume of assay medium	Stock concentration
    Oligomycin	630μL	100μM
    FCCP	720μL	100μM
    Rotenone/antimycin A	540μL	50μM

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  • b.

    Dilute the stock solutions of and Oligomycin, FCCP and Rotenone/antimycin A according to Table 2, then load 20μL of 2μM oligomycin in port A, 22μL of 1μM FCCP in port B and 25μL of 0.5μM Rotenone/antimycin A in port C of the hydrated sensory cartridge.

    Table 2

    Working concentration of oligomycin, FCCP, and rotenone/antimycin A

    Compound	Final concentration in well (μM)	Stock volume (μL)	Assay medium volume (μL)	Add to port (μL)
    Oligomycin	2.0	600	2,400	20
    FCCP	1.0	300	2,700	22
    Rotenone/antimycin A	0.5	300	2,700	25

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Note: The compounds must be prepared and loaded 20min prior to the assay.

Note: The recommended concentration of Oligomycin and Rotenone/antimycin A can meet most cell types. The maximum respiratory rate is caused by injection of uncoupler FCCP, so if OCR is not increased following the FCCP injection, you should perform a careful titration of FCCP to optimized the appropriate concentration.

• 9.

  Run assay

  • a.

    Select XF Cell Mito Stress Test on the Templates window and set up the program as follows:

  • b.

    At the Plate map page, click “add group” and select corresponding wells to edit the information of your group.

  • c.

    At the Protocol page, the parameters are as follows: baseline, 3 cycles; inject port A (oligomycin), 3 cycles; inject port B (FCCP), 3 cycles; inject port C (Rotenone/antimycin A), 3 cycles. Each cycle is composed of mix 3min, wait 0min, and measure 3min (Figure5).

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    Figure5

    The interface of a typical running protocol and experimental parameters

  • d.

    At the Run Assay page, select “Start Run” and choose a location to save your assay result file. The tray will auto-eject and put the sensory cartridge on it, the instrument will initiate the calibration of the sensory cartridge. Time to complete calibration is approximately 20min.

  • e.

    When the calibration is over, click “open the tray,” remove the utility plate and replace the XF96 Cell Culture Microplate on the tray with the correct direction as labeled on corner of the plate, which should be located at the lower-left corner of the tray, then load the tray.

Note: The default XF Cell Mito Stress Test protocol does not require modifications. The total time of OCR measurements is 1h 24min. If the readings are very slow the cells will have reduced viability which will affect the results.

CRITICAL: Before start the assay, make sure that the compounds should be injected in the port. View cells under a microscope to ensure the cell health, seeding confluence, and ensure your background wells do not contain cells.

• 10.

  Data Analysis

  • a.

    Remove the cell plate and the sensory cartridges when the run is completed.

  • b.

    Insert a USB drive and export the results.

  • c.

    Results will be automatically generated and analysis by the wave software, which can export your data as an Excel or Prism file. (https://www.agilent.com/zh-cn/product/cell-analysis/real-time-cell-metabolic-analysis/xf-software/seahorse-wave-desktop-software-740897).

Note: Desirable range of starting OCR should be between 20 and 160 pmol/O₂ /min.

Problem 1

Poor basal signal (step 10)

Potential solution

Poor basal signal could be caused by insufficient cell number and you can increase the number of cells (step 3). Another reason for poor basal signal may be incubate the cell microplate in a non-CO₂, 37°C incubator too long (step 7d). This will decrease the viability of cells. Make sure the time for incubating the cell plate in a non-CO₂, 37°C incubator is at least 30min but no more than 1 h.

Problem 2

Minimal or unexpected changes in OCR after compounds injection (step 10)

Potential solution

When the changes in OCR is minimal in a certain well, it may be because there is no drug injected to the port of the sensory cartridge, to make sure all the ports are filled with drugs after injection (after step 8). If the changes in OCR are minimal in most wells, this may be caused by the low concentration of the drugs or the poor condition of cells. View the cells under microscope before run the assay to make sure that the cells are healthy, uniform, and in a monolayer configuration. Titration of drugs’ concentration when the cells are healthy.

Problem 3

High variability between replicates (steps 3 and 4)

Potential solution

The major cause of variation might be uneven cell numbers or the sensors does not work properly.

Therefore, it is important to make sure the same volume of cells is seeded in each well of the replicates. Meanwhile, make sure all the sensors are submerged in the XF Calibrant and hydrated in the non-CO₂, 37°C incubator for 12–18 h. Make sure to use sensory cartridges and cell microplate before the expiration date.

Problem 4

Variability between wells (step 3)

Potential solution

Cells need to culture in incubator with a humidified atmosphere of 5% CO₂ at 37°C for 12–18h prior to an XF assay. Because of interventions introduced to the cells, including genetic modifications and compound treatments, the cell number might be changed during the culture. This need to be taken into account before seeding cells. One method is to test the doubling time of cells, thus choosing the optimal number of cells in different groups. Another method is to use the CyQuant cell proliferation assay (Wettmarshausen and Perocchi, 2019).

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Qiang Wan (wanqiang@sdu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Original data for figures in the paper are available upon request.

This work was supported by the National Natural Science Foundation of China (NSFC) (grant nos. 81770729, 91749111, 82070756) and Shandong Province Taishan Scholar Project (grant no. tsqn20161073).

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