Protocol for measuring respiratory function of mitochondria in frozen colon tissue from rats

Summary

Mitochondrial respirometry allows for the comprehensive study of oxygen consumption within the electron transport system in tissues. However, limited techniques exist for analyzing frozen or biobanked intestinal tissues. Here, we present a protocol to evaluate the respiratory function of mitochondria in colonic tissues after cryopreservation at −80°C. We describe steps for rat dissection, respirometry calibration, and tissue preparation. We then detail measurement of oxygen respiration and protein concentration. This protocol facilitates the retrospective analysis of mitochondrial respiration in frozen tissue.

Graphical abstract

Highlights

• •Activities of selected mitochondrial complexes are maintained post-cryopreservation
• •Frozen tissue enables retrospective analysis of tissues unavailable for immediate testing
• •Mitochondria respiration analysis facilitates knowledge of colonic metabolism

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Mitochondrial respirometry allows the comprehensive study of oxygen consumption within the electron transport system in tissues. However, limited techniques exist for analyzing frozen or biobanked intestinal tissues. Here, we present a protocol to evaluate the respiratory function of mitochondria in colonic tissues after cryopreservation at −80°C. We describe steps for rat dissection, respirometry calibration, and tissue preparation. We then detail measurement of oxygen respiration and protein concentration. This protocol facilitates the retrospective analysis of mitochondrial respiration in frozen tissue.

Before you begin

The protocol below describes the specific steps for using frozen colon tissue from adult rats (Figure 1). For fresh tissue, extra titration with saponin or digitonin may be needed to increase the cell permeability. We have also used this protocol for 2 mg frozen liver tissue from adult rats and 10 mg frozen colon tissue from adult mice. Refer to key resources table and materials and equipment sections for a complete list of necessary solutions, materials and equipment.

Figure 1.

Schematic workflow of overall study

Institutional permissions

Experimental procedures involving animals were approved by the Health Sciences Animal Care Committee at the University of Calgary under the guidelines of the Canadian Council on Animal Care.

Rat dissection

Timing: 10 min

• 1.
  Rat euthanasia by overdose inhalation of isoflurane.

  • a.
    Place rats into a clear, plastic, sealable euthanasia chamber that fits the rat size.
  • b.
    Open the cap, pour adequate isoflurane (e.g., 0.25 mL isoflurane for a 500 mL container to provide at least a 10% concentration) and close the cap.
  • c.
    Observe the rat until it stops breathing followed by decapitation.

Note: Perform euthanasia process in fume hood.

Note: Sprague-Dawley rat colonies were bred and maintained in a specific pathogen-free facility at the Health Sciences Animal Resource Centre of University of Calgary.

• 2.Dissect proximal colon. Wash out the luminal contents 3 times with saline. Snap-freeze tissue and store samples in −80°C.

Note: For samples used in the protocol, colon tissues have been stored at −80°C for 48 h or 4 months. The amount of tissue needs to be optimized to match the research purpose and detection limitation of Oxygraph-2k respirometer.

Respirometry calibration

Timing: 60 min

• 3.Prepare mitochondrial respiration MiR05¹ solution following materials and equipment section.
• 4.Calibrate the respirometry following Oxygraph-2k respirometer^1,2 user manual for specific procedures.

Note: Calibration of the Oxygraph is an essential step to minimize confounding effects of instrumental oxygen consumption.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
KOH	Sigma-Aldrich	Cat# 221473
KCl	Sigma-Aldrich	Cat# P3911
NaOH	Sigma-Aldrich	Cat# SX0590
Sucrose	Sigma-Aldrich	Cat# S0389
Mannitol	Sigma-Aldrich	Cat# M9546
KH2PO4	Sigma-Aldrich	Cat# P0662
MgCl2.6H2O	Sigma-Aldrich	Cat# M0250
EGTA	Sigma-Aldrich	Cat# E3889
HEPES	Sigma-Aldrich	Cat# H7523
Taurine	Sigma-Aldrich	Cat# T0625
Lactobionic acid	Sigma-Aldrich	Cat# L2398
Bovine serum albumin	Sigma-Aldrich	Cat# A7511
Nicotinamide adenine dinucleotide	Sigma-Aldrich	Cat# N8129
Oligomycin	Sigma-Aldrich	Cat# O4876
Succinate	Sigma-Aldrich	Cat# S2378
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)	Sigma-Aldrich	Cat# C2920
Rotenone	Sigma-Aldrich	Cat# R8875
Antimycin A	Sigma-Aldrich	Cat# A8674
Sodium dithionite	Merck KGaA	Cat# 106507
Saline	Cytiva	Cat# Z1377
HCl	VWR	Cat# 46414-320
EtOH	Greenfield Global	Cat# P006EAAN
Isoflurane	Fresenius Kabi	Cat# CP0406V2
Ammonium solution 25%	Supelco	Cat# 105432
Polishing Powder 0.3 μm	Oroboros Instruments	Cat# 26520-01
Experimental Models: Organisms/Strains
Rattus norvegicus: Sprague-Dawley 11-week-old rat (males)	In-house breeding	Strain code: 400; RRID: MGI:5651135
Critical commercial assays
BCA Protein Assay Kit	Thermo Scientific	Cat# 23225
Software and Algorithms
DatLab 4	Oroboros Instruments	N/A
BioRender	BioRender	N/A
Other
Oxygraph-2k respirometer	Oroboros Instruments	Product# 10033-01
Model 1702 N microsyringe 25 μL	Hamilton	Cat# 80275/00
Model 1701 N microsyringe 10 μL	Hamilton	Cat# 148151
pH meter	Mettler Toledo	FiveEasy pH meter F20
Teflon-glass homogenizer	Wheaton	Cat# 14-6096-28
0.22 μm cellulose nitrate filter system	Corning	Cat# 430758
OroboPOS-Membranes	Oroboros Instruments	Product# 26200-01
96-well plate	Corning	Cat# 351172
ELISA microplate reader	BioTek	Synergy HT
Eppendorf 5424R microcentrifuge	Eppendorf	Cat# 5406000119
Surgical Scissors	FST	Cat# 14002-12
1.5 mL Microcentrifuge tube	Axygen	Cat# MCT-150-A
50 mL Conical tube	Falcon	Cat# 352070
37°C Incubator	VWR	Cat# 97025-630
−80°C Freezer	Revco	Model# ULT2586-5-A39

Materials and equipment

• •0.5 M Potassium-lactobionate Stock: Add 35.83 g lactobionic acid to 100 mL ddH₂O and adjust pH to 7.0 with 5 M KOH at 21°C–25°C. Adjust volume to 200 mL with ddH₂O. Prepare the fresh stock before use.
• •Mitochondrial respiration buffer MiR05.

Reagent	Final concentration	Amount
EGTA	0.5 mM	0.190 g
MgCl2.6H2O	3.0 mM	0.610 g
Taurine	20.0 mM	2.502 g
KH2PO4	10.0 mM	1.361 g
HEPES	20.0 mM	4.77 g
Potassium-lactobionate	60.0 mM	120 mL of 0.5 M Potassium-lactobionate
Sucrose	110.0 mM	37.65 g
Bovine serum albumin	1 g/L	1 g
ddH2O	N/A	∼1,000 mL
Total	N/A	1,000 mL

Note: Adjust pH to 7.1 with 5 M KOH at 30°C, filter with a 0.22 μm bottle0top filter, dispense into 50 mL aliquots and store at −20°C for up to 6 months.

• •Mitochondrial assay buffer.

Reagent	Final concentration	Amount
Sucrose	70.0 mM	23.96 g
MgCl2.6H2O	5.0 mM	1.017 g
Mannitol	220.0 mM	40.0 g
KH2PO4	5.0 mM	0.68 g
EGTA	1.0 mM	0.380 g
HEPES	2.0 mM	0.477 g
ddH2O	N/A	∼1,000 mL
Total	N/A	1,000 mL

Note: Adjust pH to 7.4 with 5 M KOH, filter with a 0.22 μm bottle-top filter, and dispense into 50 mL aliquots and store at −20°C for up to 6 months.

• •10 mM NaOH: Dissolve 40 mg NaOH in 100 mL ddH₂O. Store at 21°C–25°C for up to 6 months.
• •10 mM NADH: Dissolve 7.1 mg NADH in 1 mL 10 mM NaOH. Store at −20°C for up to 3 months.
• •4 mg/mL Oligomycin: Dissolve 4 mg Oligomycin in 1 mL 100% ethanol. Store at −20°C in 250 μL aliquots for up to 3 months.
• •1 M Succinate: Dissolve 1.3505 g succinate in 3 mL ddH₂O. Adjust pH with 1 M HCl to a final volume of 5 mL. Store at −20°C in 250 μL aliquots for up to 3 months.
• •0.1 mM Rotenone: Dissolve 0.39 mg rotenone in 10 mL 100% ethanol. Store at −20°C in 250 μL aliquots for up to 3 months.
• •2 mM Antimycin A: Dissolve 10.96 mg Antimycin A in 10 mL 100% ethanol. Store at −20°C in 250 μL aliquots for up to 3 months.
• •1 M KCl: Dissolve 74.55 g of KCl in ddH₂O to a final volume of 1 L. Store at 21°C–25°C for up to 6 months.
• •5 M KOH: Dissolve 14.028 g of KOH in ddH₂O to a final volume of 50 mL. Store at 21°C–25°C for up to 6 months.
• •1 M HCl: Dissolve 98.9 mL of 37% HCl solution in ddH₂O to a final volume of 1 L. Store at 21°C–25°C for up to 6 months.

Step-by-step method details

Calibrate machine for respirometry

Timing: 60 min

Respirometry calibration is the major step and prerequisite to obtain accurate measurement of oxygen respiration.

• 1.Open DatLab software³ and connect to Oxygraph-2k (Figure 2).
• 2.Rinse chambers with 3 cycles of water and 100% ethanol. Leave the chamber dry enough for 15 min.

Note: Avoid touching the polarographic oxygen sensor inside the chamber during suctioning washout and titrating.

• 3.Fill the chamber with 2.4 mL pre-warmed (37°C) MiR05.

Note: Keep MiR05 in 37°C during the test to reduce bubbles introduced from environment to the chamber.

• 4.Follow the standard Air and zero calibration protocol for Oxygraph. Refer to https://wiki.oroboros.at/index.php/Oxygen_calibration_-_DatLab for details and troubleshooting (Figure 3).

Note: Zero voltage should not exceed 5% of the voltage at air saturation procedure to achieve calibration.

• 5.After calibration, rinse chambers with 3 cycles of water and 100% ethanol. After drying, fill the chamber with 2.4 mL pre-warmed MiR05. Close the insert. The Oxygraph is ready for use now.

Figure 2.

The Oxygraph-2k Respirometer system

Figure 3.

Trace of Oxygraph calibration

Tissue preparation

Timing: 30 min

Colon tissue was collected from the adult rat, and stored at −80°C. We recommend tissue be snap-frozen in liquid nitrogen to reserve mitochondrial activity.

• 6.Weigh 50 mg frozen tissues in 500 μL ice-cold mitochondria assay buffer. Cut tissues into ∼10 pieces.

Note: Cutting into pieces helps with the homogenization of the tissue.

• 7.Homogenize with 15–20 stokes in Teflon-glass homogenizer.^4,5 Transfer all homogenate into a clean 1.5 mL Eppendorf tube (Figure 4).
• 8.Centrifuge homogenate at 1,000 g for 15 min at 4°C.

Note: Low temperature is essential to reserve mitochondria activity.

Figure 4.

Overview of sample preparation steps

Measure oxygen respiration

Timing: 60 min

This step is core to analyze mitochondrial bioenergetics. Mitochondrial oxygen respiration will be detected with Oxygraph-2k.

• 9.Add 100 μL supernatant with pipette to Oxygraph chamber. Close the chamber. Wait for 5–10 min until the curve of oxygen consumption rate (red curve) is stable.

Note: Avoid pipetting solid tissue residues into the chamber.

CRITICAL: Supernatant amount is important to measure oxygen respiration. Excess or insufficient amounts could trigger improper mitochondrial responses. The amount should be optimized for other tissue types. For example, a different amount is used for skeletal muscle.⁶ For mice, 10 mg of frozen colon tissue is recommended based on previous testing in our laboratory.

• 10.Titrate 10 μL NADH solution to the chamber (final concentration, 0.05 mM) (Figure 5). Record 3–5 min of stable Complex I-linked respiration.

CRITICAL: Carefully clean Hamilton syringe three times with 100% EtOH and ddH₂O after each injection.

CRITICAL: Store substrates and inhibitors on ice and protect from light during the day of experiment.

• 11.Titrate 1 μL Oligomycin to the chamber (final concentration, 2 μg/mL). Record 3–5 min of stable ATP production-linked respiration.

Note: The concentration for substrate and inhibitor stocks refers to those concentrations for brain^7,8,9 and liver tissues.¹⁰

• 12.Titrate 20 μL succinate to the chamber (final concentration, 10 mM). Record 3–5 min of stable Complex II-linked respiration.
• 13.Step-wise titration of 1 μL FCCP to the chamber until achieving the stable maximal respiration.

Note: FCCP is light sensitive. It decomposes and loses its activity when exposed to light. Handle and store rotenone in dark conditions such as aluminum foil to preserve activity.

CRITICAL: FCCP is harmful if swallowed. It causes severe skin burns and eye damage and may cause skin allergy. In case of eye or skin contact, wash thoroughly immediately with plenty of water.

• 14.Titrate 1 μL rotenone to the chamber (final concentration, 0.05 μM). Record 3–5 min of stable Complex IV-linked respiration.

Note: Rotenone is light sensitive. It decomposes and loses its activity when exposed to light. Handle and store rotenone in dark conditions such as aluminum foil to preserve activity.

CRITICAL: Rotenone is fatal if swallowed and inhaled. It causes skin irritation, serious eye irritation and may cause respiratory irritation. In case of eye or skin contact, wash thoroughly immediately with plenty of water.

• 15.Titrate 2.5 μL Antimycin A to the chamber (final concentration, 2.5 μM). Record 3–5 min of residual non-mitochondria respiration.

CRITICAL: Antimycin A is fatal if swallowed.

• 16.Collect all of the solution in the chamber to a 2 mL Eppendorf tube.
• 17.Wash the chamber with 3 cycles of water and 100% ethanol to remove EtOH-soluble inhibitors. Dry the chamber totally for 20–30 min before testing next sample.

Note: Please wash more times if there is residue left in the chamber.

Figure 5.

Titrate substrates/inhibitors

Measure protein concentration

Timing: 60 min

This step measures protein concentration in supernatant for normalizing oxygen respiration.

• 18.Measure the protein concentration in supernatant collected in step 8 using the Pierce BCA Protein Assay Kit (Figure 6) for normalization purposes.

Note: Other BCA protein assay kits or reliable assays can also be used to determine total protein concentration of the assessed sample.

• 19.Put the concentration value in DatLab to normalize the value of oxygen respiration rate.

Figure 6.

Linear standard curve from BCA assay

Expected outcomes

This method is employed to measure mitochondrial respiration in frozen colon tissue. The activity of mitochondria complex I and II, as well as maximal respiration, is preserved after cryopreservation as shown by the expected increase in oxygen consumption rate following NADH and succinate titration. The use of frozen tissue provides more flexibility when measurement is not possible for multiple fresh tissues. A representative Oxygraph curve is shown in Figure 7. An example comparison Oxygraph of results from fresh and frozen tissue is shown in Figure 8. The values from fresh tissue are slightly lower than frozen tissue, which may be due to freezing tissue that results in cell lysis allowing increased membrane permeability to both inhibitors and substrates. When using fresh tissue with the present protocol, membrane permeabilizers such as saponin or digitonin are needed to increase the cell permeability. Levels of permeabilizers need to be optimized prior to starting experimentation.¹¹

Figure 7.

A representative trace of mitochondrial respiration in frozen colon tissue after 4-month cryopreservation

Figure 8.

Oxygen consumption in fresh and frozen tissues

(A and B) Trace plots of fresh (A) and frozen tissue after cryopreservation for 48 h (B).

Quantification and statistical analysis

It is recommended to use manufacturer software for data analysis (DatLab for Oroboros O2k). Use the following formula to calculate the final value of oxygen consumption rate. An example of oxygen flux readouts from sample shown in Figure 7 is present in Figure 9.

$$\mathrm{C}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{x}\mathrm{I}-\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{k}\mathrm{e}\mathrm{d}\mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left(\mathrm{O}\mathrm{x}\mathrm{y}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{u}\mathrm{m}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{N}\mathrm{A}\mathrm{D}\mathrm{H}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right)-(\mathrm{N}\mathrm{o}\mathrm{n}-\mathrm{m}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{c}\mathrm{h}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n})$$

$$\mathrm{A}\mathrm{T}\mathrm{P}\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left(\mathrm{O}\mathrm{x}\mathrm{y}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{u}\mathrm{m}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{N}\mathrm{A}\mathrm{D}\mathrm{H}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right)-\left(\mathrm{M}\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{m}\mathrm{a}\mathrm{l}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{O}\mathrm{l}\mathrm{i}\mathrm{g}\mathrm{o}\mathrm{m}\mathrm{y}\mathrm{c}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right)$$

$$\mathrm{C}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{x}\mathrm{I}\mathrm{I}-\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{k}\mathrm{e}\mathrm{d}\mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left(\mathrm{O}\mathrm{x}\mathrm{y}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{u}\mathrm{m}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{S}\mathrm{u}\mathrm{c}\mathrm{c}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right)-(\mathrm{N}\mathrm{o}\mathrm{n}-\mathrm{m}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{c}\mathrm{h}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n})$$

$$\mathrm{M}\mathrm{a}\mathrm{x}\mathrm{i}\mathrm{m}\mathrm{a}\mathrm{l}\mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left(\mathrm{O}\mathrm{x}\mathrm{y}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{u}\mathrm{m}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{F}\mathrm{C}\mathrm{C}\mathrm{P}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right)-(\mathrm{N}\mathrm{o}\mathrm{n}-\mathrm{m}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{c}\mathrm{h}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n})$$

Figure 9.

Oxygen flux of the sample present in Figure 7

Limitations

In this protocol, 50 mg of frozen colon tissue was examined. The tissue amount needs to be determined for each tissue types such as cardiac fibers,¹ hippocampus^8,12,13 and epithelial cells.² The method is not able to measure proton leak-related respiration. In this protocol, tissues frozen for 48 h or 4 months were used. However, mitochondrial activity may drop in samples after longer-term cryopreservation. The capacity of Oroboros respirometry is limited by the number of chambers per run (1 sample/chamber). Other platforms, such as Agilent Seahorse XF analyzer, could be employed for large batches of samples that need to be tested simultaneously.^5,14

Troubleshooting

Problem 1

The zero voltage during calibration is higher than 5% of the voltage obtained at air saturation (related to “step-by-step method details-calibrate machine for respirometry” section, steps 1–5).

Potential solution

Service the polarographic oxygen sensor following protocol at https://wiki.oroboros.at/index.php/Polarographic_oxygen_sensor and calibrate again following the protocol at https://bioblast.at/images/archive/d/d4/20140216163427!MiPNet19.01B_POS-Service.pdf.

Disconnect the polarographic oxygen sensor.

Clean anode by 25% ammonium solution and cathode by polishing powder.

Mount a new oxygen sensor membrane.

Note: Avoid introducing any bubble when mounting a new membrane.

Problem 2

After centrifuging tissues, a white layer is present on the surface of the homogenate (related to “step-by-step method details-tissue preparation” section, steps 6–8).

Potential solution

This may be caused by the mesenteric adipose in tissue. When dissecting animals, carefully remove the surrounding adipose with forceps and surgery scissors.

Problem 3

The Oxygraph curve is not stable with large oscillations before and/or during test (related to “step-by-step method details-measure oxygen respiration” section, steps 9–15).

Potential solution

This may be caused by extra bubbles on top of the polarographic oxygen sensor. To solve the issue, stop the test and service the polarographic oxygen sensor until a stable trace curve achieved.

Problem 4

Mitochondria fails to respond properly after NADH, Oligomycin, Succinate and FCCP titration (related to “step-by-step method details-measure oxygen respiration” section, steps 10–15).

Potential solution

This may be caused by three reasons. First, the tissue may have lost activity due to improper processing (e.g., homogenization) or storage. This may be solved by testing a new piece of tissue. Second, this can be caused by residual inhibitors in the respirometry chamber from previous titration. This can be solved by washing the Oroboros chamber totally with 100% ethanol and water. Another possible reason may be the degradation of substrate stock. This can be solved by preparing a new batch of stock solution.

Problem 5

Low flux values (e.g., <10 pmol/(s·mg)) across titrations for a batch of samples (related to “step-by-step method details-measure oxygen respiration” section, steps 10–15).

Potential solution

This may be caused by failure of the polarographic oxygen sensor. Service the polarographic oxygen sensor following the protocol at https://bioblast.at/images/archive/d/d4/20140216163427!MiPNet19.01B_POS-Service.pdf.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chunlong Mu(chunlong.mu1@ucalgary.ca).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate any unique datasets or code.