Protocol for measuring mitochondrial respiratory capacity from buccal cell swabs

Summary

Mitochondrial respirometry provides a detailed assessment of oxygen consumption within the electron transport system, yet methods detailing respiration from non-invasive samples remain limited. Here, we present a protocol for measuring mitochondrial respiration in cultured buccal cells. We outline procedures for buccal cell collection, primary cell culture, and respirometry calibration, followed by oxygen consumption measurements and cell count for data normalization. This protocol allows reliable evaluation of mitochondrial function from non-invasive buccal cell samples, offering a valuable tool for metabolic investigation.

Graphical abstract

Highlights

• •Steps for culturing primary buccal cells to be used for detailed respirometry
• •Instructions for measuring mitochondrial complex activity using cultured buccal cells
• •Buccal cell analysis provides a non-invasive proxy for mitochondrial bioenergetics

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

Mitochondrial respirometry provides a detailed assessment of oxygen consumption within the electron transport system, yet methods detailing respiration from non-invasive samples remain limited. Here, we present a protocol for measuring mitochondrial respiration in cultured buccal cells. We outline procedures for buccal cell collection, primary cell culture, and respirometry calibration, followed by oxygen consumption measurements and cell count for data normalization. This protocol allows reliable evaluation of mitochondrial function from non-invasive buccal cell samples, offering a valuable tool for metabolic investigation.

Before you begin

This protocol describes the specific steps to measure mitochondrial bioenergetics using cultured buccal cell samples (Figure 1). Buccal cell swabs were selected for their non-invasive collection allowing it to be suitable for use in pediatric studies. For details on this protocol, please refer to key resources table and materials and equipment for all necessary materials to complete the following assays.

Figure 1.

Schematic workflow for buccal cell respirometry

Institutional permissions

Experimental procedures involving human cell samples were approved by the Research Ethics Board at the University of Calgary (REB# 23-1557).

Buccal cell collection and preparation for culture

Timing: 10 min for collection, 1 h for initial processing, ongoing for culture (4–7 days)

• 1.
  Buccal Cell Collection (see Figure 2).

  • a.
    Place Mitochondrial respiration buffer, MiR05 in 37°C incubator (VWR, see key resources table) for 1 h prior to collection.
  • b.
    Use a sterile nylon-flocked or cotton swab to firmly swab the inner cheek for 10 s, ensuring adequate cell collection.
  • c.
    Immediately immerse the swab in a sterile tube containing MiR05 respiration buffer (at least 1 mL).
  • d.
    Vortex the tube gently for 10 s to dislodge cells from the swab.
  • e.
    Remove the swab and discard it in a biohazard waste container.
  • f.
    Store the sample at −20°C or −80°C until further processing. If long term (> 24 h and up to a year), using 10% Dimethylsulfoxide (DMSO).¹,² Store the sample at −80°C until further processing. If long term (>24 h), use DMSO.

    • i.
      If using sample immediately, skip freezing and proceed to step 2a.

Note: Rapid freezing helps preserve mitochondrial function for subsequent respirometry measurements.³ However, freezing cells in MiR05 for long term is not recommended and can decrease cell viability. If freezing cells for long periods of time, store in DMSO.^4,5 If able to culture immediately, skip freezing and place freshly swabbed cells in MiR05 at 37°C.

• 2.
  Thawing and Preparation for Culture.

  • a.
    Retrieve the frozen sample from −80°C storage and allow it to thaw at room temperature for ∼5 min or directly in 37°C water bath.

    • i.
      This applies for both cells frozen in MiR05 and DMSO.
  • b.
    Transfer the thawed suspension to a sterile 15 mL conical tube.
  • c.
    Pass the cell suspension through a 40–80 μm cell strainer into a new sterile tube to remove debris and contaminants. A 40 μm cell strainer was used in the present protocol.
  • d.
    Centrifuge the filtered sample at 300 × g for 5 min at room temperature to pellet the buccal cells.
  • e.
    Carefully remove the supernatant and resuspend the cell pellet in 5 mL of pre-warmed complete growth medium (Dulbecco’s Modified Eagle Medium, DMEM + 10% Fetal Bovine Serum, FBS).

    • i.
      Prewarm medium in a water bath at 37°C for 30 min.
    • ii.
      Complete medium can be prepared beforehand and stored at 4°C for up to six weeks.

Note: Using larger cell strainers is possible, but may increase the presence of debris and contaminants. The goal is not to capture every cell, but to obtain a sufficient number for successful cell culturing.

• 3.
  Primary Buccal Cell Culture.

  • a.
    Transfer the resuspended cells to a T25 tissue culture flask.
  • b.
    Incubate cells at 37°C with 5% CO₂ in a humidified incubator.
  • c.
    For the first 24 h, supplement the growth medium with 1% antibiotic-antimycotic solution (60 μL of antibiotic solution for 6 mL DMEM in T25 flask) to prevent bacterial contamination. Other antibiotic-antimycotic solutions can be used at appropriate concentrations.
  • d.
    After 24 h, replace the medium in the T25 flask with complete growth medium, without antibiotics (DMEM + 10% FBS) to promote cell viability.
• 4.
  Culture Maintenance.

  • a.
    Monitor cells daily for adherence and morphology (round and large cytoplasm to nucleus ratio) under an inverted microscope (Figure 3).
  • b.
    Change the medium every 48 h, carefully aspirating old medium and replacing it with fresh pre-warmed medium.
  • c.
    Once cells reach ∼80% confluency (∼5 – 7 days; maximum passage of 3 times), they can be used for mitochondrial respirometry analysis.

Note: Check for viability using any cell counter.⁶

Note: Optimization of culture conditions, including medium composition and substrate supplementation, may be necessary depending on experimental goals.

Figure 2.

Overview of sample preparation

Figure 3.

Trypan blue stain imaging of buccal cell for morphology and health

(A) Representative image of healthy buccal cells displaying round, ovular morphology and (B) unhealthy cells are shrunken irregular morphology and greater trypan blue penetration.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Potassium hydroxide (KOH)	Sigma-Aldrich	Cat# 221473
Potassium chloride (KCl)	Sigma-Aldrich	Cat# P3911
Sodium hydroxide (NaOH)	Sigma-Aldrich	Cat# SX0590
Sucrose	Sigma-Aldrich	Cat# S0389
Mannitol	Sigma-Aldrich	Cat# M9546
Potassium dihydrogen phosphate (KH2PO4)	Sigma-Aldrich	Cat# P0662
Magnesium chloride hexahydrate (MgCl2.6H2O)	Sigma-Aldrich	Cat# M0250
Ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (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
Oligomyicn	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
Sodium azide	Sigma-Aldrich	Cat# S2002
Hydrochloric acid (HCl)	VWR	Cat# 46414-320
Ethanol (EtOH)	Greenfield Global	Cat# P006EAAN
Ammonium solution 25%	Supelco	Cat# 105432
Polishing powder 0.3 μm	Oroboros Instruments	Cat# 26520-01
Phosphate-buffered saline 10× (PBS)	Thermo Scientific	Cat# 70011044
Dulbecco’s modified Eagle’s medium (DMEM)	Thermo Scientific	Cat# 11965118
Trypsin 25%, phenol red	Thermo Scientific	Cat# 25200072
Trypan blue, 0.4%	Thermo Scientific	Cat# 15250061
Fetal bovine serum (FBS)	Thermo Scientific	Cat# A5256701
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat# D4540
Antibiotic-antimycotic (100×)	Thermo Scientific	Cat# 15240062
Experimental models: Organisms/strains
Human buccal cells	Study participants	N/A
Software and algorithms
DatLab 4	Oroboros Instruments	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
0.22 μm cellulose nitrate filter system	Corning	Cat# 430758
10 mL serological pipette	Fisher Scientific	Cat# 13-675-20
Motorized pipet fillers	Thermo Scientific	Cat# 9501
OroboPOS membranes	Oroboros Instruments	Product# 26200-01
Falcon 15 mL conical centrifuge tubes	Fisher Scientific	Cat# 14-959-53A
Sterile flocked swabs	Fisher Scientific	Cat# 22-025-192
Falcon 40 μm cell strainer	Corning	Cat# 352340
1.5 mL microcentrifuge tube	Axygen	Cat# MCT-150-A
Luna-II automated cell counter	Logos Biosystems	Cat# L40002
T25 flask	Thermo Scientific	Cat# 166371
50 mL conical tube	Falcon	Cat# 352070
CO2 incubator	Thermo Scientific	Cat# 381
37°C incubator	VWR	Cat# 97025-630
Vertical laminar airflow workstation	NuAire	Cat# NU-140
Precision 182 general purpose water bath	Artisan Technology Group	Cat# 59812-3
Nikon TMS inverted phase contrast microscope	Microscope Central	Cat# TMS-M
Beckman GS-6 centrifuge	Marshall Scientific	Cat# BE-GS6
Vortex-Genie 2 mixer	Fisher Scientific	Cat# 50728002
−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 H₂O 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.

  • ○
    Store at room temperature (21°C–25°C) for up to six months. After which place in aliquots at −20°C for up to 3 months.

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 0.45 mm bottle top 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 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.
• •5 mM Pyruvate: Dissolve 55.02 mg Pyruvate in 100 mL ddH₂O. Store at −20°C in 250 μL aliquots for up to 3 months.
• •2 mM Malate: Dissolve 26.82 mg Malate in 100 mL ddH₂O. Store at −20°C in 250 μL aliquots for up to 3 months.
• •10 mM Glutamate: Dissolve 147.13 mg Glutamate in 100 mL ddH₂O. Store at −20°C in 250 μL aliquots for up to 3 months.
• •2.5 mM Adenosine Diphosphate (ADP): Dissolve 106.8 mg ADP in 100 mL ddH₂O. Store at −20°C in 250 μL aliquots 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.

  • ○
    Due to light sensitivity, store in an amber tube or wrap with aluminum foil.
• •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.
• •
  1 μM FCCP: Dissolve 254.18 mg FCCP in 1 mL 100% ethanol. Store at −20°C in 250 μL aliquots for up to 3 months.

  • ○
    Due to light sensitivity, store in an amber tube or wrap with aluminum foil.
• •
  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.

  • ○
    Due to light sensitivity, store in an amber tube or wrap with aluminum foil.
• •
  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.

  • ○
    Due to light sensitivity, store in an amber tube or wrap with aluminum foil.
• •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.
• •10 mM Sodium Azide: Dissolve 65.01 g Sodium Azide in 100 mL of ddH₂O. at −20°C in 250 μL aliquots 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. The calibration follows the method published previously.³ Refer to Figure 4 for respirometer schematic.

• 1.Open DatLab software⁷ and connect to Oxygraph-2k. Refer for user manual for specific details.⁸,⁹
• 2.Rinse chambers with 3 cycles of water and 100% ethanol. Leave the chamber dry enough for 15–20 min.

Note: Avoid touching the polarographic oxygen sensor during suctioning washout from the chamber.

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

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

• 4.Follow the standard Air and Zero calibration protocol at https://wiki.oroboros.at/index.php/Oxygencalibration-DatLab for details and troubleshooting.⁹

Note: The zero voltage should not exceed 5% of the voltage measured during air saturation procedure to ensure proper calibration.

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

Figure 4.

Respirometer schematic

Suspension of cultured cells

Timing: 60 min

• 6.Retrieve the cultured cells.
• 7.Wash cells with 1× PBS by adding 6 mL PBS to T25 flask and gently swirling the plate.
• 8.Aspirate PBS from flask and be careful not to disturb cells.
• 9.Add 3–5 mL of 0.25% Trypsin to detach cells. Incubate at 37°C for 2–5 min (monitor under a microscope for detachment).
• 10.Gently tap the flask to aid detachment.
• 11.Once detached, neutralize trypsin using 6 mL of DMEM+FBS.
• 12.Resuspend cells by gently pipetting up and down 5–10 times to break up cell clumps and ensure a homogeneous suspension.

Note: Use a serological pipette for large volumes or a P1000 pipette for smaller volumes.

• 13.Transfer cells to a 1 5 mL conical tube.
• 14.Centrifuge at 300 × g for 5 min and carefully aspirate the supernatant.
• 15.Wash cells with PBS by gently pipetting up and down with 5 mL PBS.
• 16.Centrifuge at 300 × g for 5 min and carefully aspirate the supernatant.
• 17.Repeat steps 15–16 a second time.
• 18.Remove supernatant and resuspend the pellet in 2 mL MiR05.

Measure the total cell count and viability

Timing: 10 min

• 19.Gently resuspend the cells in the culture medium by pipetting up and down to ensure an even distribution.
• 20.If the cell concentration appears too high (> 1 × 10⁵ cells),¹⁰ dilute the sample with an appropriate buffer (e.g., PBS or culture medium) to fall within the optimal counting range (1.0 × 10² cells per 1 mm²).
• 21.In a microcentrifuge tube, mix 10 μL of cell suspension with 10 μL of 0.4% Trypan Blue (1:1 dilution). Gently pipette up and down to mix.
• 22.Load 10 μL of the mixed sample into one of the chambers of the LUNA II counting slide. Ensure there are no bubbles in the chamber.
• 23.Insert the slide into the LUNA II automated cell counter and run the cell count according to device instruction.
• 24.Record the cell count for normalization of respiration values. Enter values in DatLab to normalize the value of oxygen respiration rate.

Figure 5 outlines the critical steps for cell counting. This step ensures accurate quantification of viable cells (refer to Figure 6), which is critical for downstream applications such as respirometry, seeding for culture, or experimental standardization.

Figure 5.

Counting cells using automated cell counter and trypan blue staining

Data are the mean ± SD, n=3.

Figure 6.

Viability and cell count data

Data are the mean ± SD, n=3.

Measure oxygen respiration

Timing: 60 min

This step is pertinent to analyze mitochondrial bioenergetics by utilizing Oxygraph-2k.

• 25.Transfer 2 mL of the suspended culture with pipette to Oxygraph chamber. Securely close the chamber lid. Wait for 5–15 min until the curve of oxygen consumption rate (red curve) is stable.

Note: The total volume added to the chamber affects the accuracy of oxygen consumption. Using too little or too much may result in atypical mitochondrial energetics. Our lab has previously determined the amount for other tissue types including skeletal muscle,¹¹ and hippocampal tissue,³,¹² liver and prefrontal cortex.¹³,¹⁴

• 26.Inject 0.5 μL Digitonin into the chamber. Record permeabilized cell respiration for 3–5 min.

Note: Thoroughly clean Hamilton syringe three times alternating between 100% EtOH and H₂O after each injection.

• 27.Sequentially inject, 5, 2.5, 10 μL of Pyruvate, Malate and Glutamate solution respectively to the chamber. Record stable Complex I-linked respiration for 3–5 min.
• 28.Inject 10 μL ADP into the chamber. Record 3–5 min of oxidative phosphorylation-stimulated respiration.
• 29.Inject 5 μL Cytochrome c into the chamber. Evaluate outer mitochondrial membrane integrity for 3–5 min.
• 30.Inject 1 μL Oligomycin into the chamber. Record ATP synthesis-coupled respiration, record for 3–5 min.
• 31.Inject 20 μL succinate into the chamber. Record Complex II-driven respiration for 3–5 min.
• 32.Inject 1 μL FCCP in a step-wise manner to the chamber until achieving the stable maximal respiration.

Note: FCCP is light sensitive. Protect from light using aluminum foil or dark storage conditions.

• 33.Inject 1 μL rotenone into the chamber. Record Complex IV-linked respiration for 3–5 min.

Note: Rotenone is light sensitive. Protect from light using aluminum foil or dark storage conditions.

• 34.Inject 50 μL Sodium Azide into the chamber. Record 3–5 min of residual non-mitochondria respiration.
• 35.Transfer contents of chamber into a 2 mL Eppendorf tube.
• 36.Rinse the chamber three times with water and 100% ethanol to remove residual inhibitors. Air dry completely for 30 min before running next sample.

Expected outcomes

This protocol enables the of mitochondrial respiration in buccal cell samples. This allows investigators to determine mitochondrial function of individuals from non-invasive means of sample collection. Mitochondrial function, including the activity of Complex I and II and maximal respiratory capacity, is preserved following cryopreservation, and culturing as demonstrated by the expected increase in oxygen consumption rate after sequential substrate titration. The use of frozen buccal cells provides greater flexibility in sample processing, allowing for batched measurements when immediate respirometry analysis is not feasible and given the small number of cells collected. A representative Oxygraph trace of buccal cell respiration is shown in Figure 7.

Figure 7.

Representative trace of mitochondrial respiration from buccal cell culture suspension

Quantification and statistical analysis

It is recommended to use manufacturer software to data analysis (DatLab for Oroboros O2k – Oxygraph-2k). Use the following formula to calculate the final value of oxygen consumption rate as shown in Figure 8.

$$\text{Complex}\mathrm{I}\text{Phosphorylation}\text{Respiration}=\left(\text{Oxygen}\text{consumption}\text{rate}\text{after}\text{ADP}\text{titration}\right)-\left(\text{Non}⎼\text{mitochondrial}\text{Respiration}\right)$$

$$\text{Complex}\mathrm{I}\text{Proton}\text{Leak}\text{Respiration}=\left(\text{Oxygen}\text{consumption}\text{rate}\text{after}\text{PMG}\text{titration}\right)-\left(\text{Non}⎼\text{mitochondrial}\text{Respiration}\right)$$

$$\text{Complex}\mathrm{I}+\text{II}\text{Phosphorylation}\text{Respiration}=\left(\text{Oxygen}\text{consumption}\text{rate}\text{after}\text{Succinate}\text{titration}\right)-\left(\text{Non}⎼\text{mitochondrial}\text{Respiration}\right)$$

$$\text{Maximal}\text{Respiration}\text{of}\text{Electron}\text{Transport}\text{Chain}=\left(\text{Oxygen}\text{consumption}\text{rate}\text{after}\text{FCCP}\text{titration}\right)-\left(\text{Non}⎼\text{mitochondrial}\text{Respiration}\right)$$

Figure 8.

Mitochondrial bioenergetics

Data are the mean ± SD, n=3.

Limitations

In this protocol, buccal cells were collected, cryopreserved, and cultured for mitochondrial respirometry analysis. Because cell yield can be influenced by factors such as donor variability, collection efficiency and culture conditions, the optimal number of cells required for accurate respirometry measurements must be determined experimentally. In order to preserve viability, buccal cell samples were frozen for up to 24 h prior to culturing; although cells remained viable within this time frame (Figure 5), prolonged cryopreservation may lead to a decline in mitochondrial activity.¹,⁴,⁵ Given that cells collected directly from swabs are limited in number, culturing is necessary to increase yield for detection in the Oroboros O2K respirometer. Other respirometry systems, including the Seahorse XF Analyzer (Agilent, Santa Clara, CA) also exist for the assessment of mitochondrial function. Buccal cell measures may be possible on other systems, but would require further, system specific experimental optimization. Future studies should further investigate the stability of mitochondrial function in buccal cells following longer-term storage.

Troubleshooting

Problem 1

Primary buccal cell cultures show low viability after thawing from cryopreservation.

Potential solution

Ensure cells are cryopreserved in an appropriate freezing mediums (e.g., 10% DMSO for long term storage or in MiR05 (up to 24 h).² If culturing immediately after collection, ideally do not freeze samples, keep in MiR05 at 37°C.

Problem 2

The use of Antimycin A as a Complex III inhibitor leads to an increase in reactive oxygen species (ROS) production, which may interfere with accurate respirometry measurements.¹⁵

Potential solution

To minimize ROS-related artifacts, replace Antimycin A with sodium azide, which inhibits Complex IV while reducing ROS generation. Ensure proper titration of sodium azide to avoid excessive inhibition that may affect mitochondrial respiration measurements.

Problem 3

Contamination frequently occurs in cultured buccal cells.

Potential solution

Use strict aseptic techniques, including working in a biosafety cabinet, using filtered pipette tips, and regularly sterilizing work surfaces. Additionally, supplement media with antibiotics/antimycotics (e.g., 1% penicillin-streptomycin) and monitor contamination through routine microscopy.

Note: Do not incubate for longer than 24 h as antibiotics have been shown to affect mitochondrial function. Keep monitoring under microscope for any sudden changes in morphology.¹⁶

Problem 4

Some buccal cell samples do not show an expected increase in oxygen consumption following saponin treatment for membrane permeabilization.

Potential solution

In cases where saponin is insufficient for effective permeabilization, digitonin can be used as an alternative due to its higher hydrophobicity, which allows more efficient permeabilization of lipid-enriched samples.¹⁷ Optimize digitonin concentration empirically to ensure selective permeabilization without excessive mitochondrial damage.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jane Shearer (jshearer@ucalgary.ca).

Technical contact

Questions regarding then technical details of the protocol should be directed to the technical contact, Tina Ram (tina.ram@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.

Acknowledgments

This study was funded by the Natural Sciences and Engineering Research Council of Canada (J.S., RGPIN-2018-04238), and T.R.R. received graduate funding from the Canadian Institutes of Health Research-funded Canadian Graduate Scholarship – Masters (CIHR CGSM).

Author contributions

T.R.R., C.M., S.J.M., and J.S. conceived, designed, and supervised the project. T.R. conducted most of the experiments and performed data analysis. J.C.C. contributed to data collection and testing.

Declaration of interests

The authors declare no competing interests.