Protocol for investigating mitochondrial structure, function, and metabolism in human cervical cancer cells

Summary

Mitochondria regulate a variety of biological activities, including metabolism, oxidative stress, and cell death. Here, we present a protocol for the investigation of mitochondrial structure, function, and metabolism in human cervical cancer cells. We describe steps for staining and visualizing mitochondria using confocal microscopy to assess morphology, mass, membrane potential, calcium, reactive oxygen species (ROS), and lipid droplet accumulation. We then detail procedures for isolating mitochondria and performing metabolomic analysis of mitochondrial metabolites via mass spectrometry.

For complete details on the use and execution of this protocol, please refer to Adiga et al.¹

Graphical abstract

Highlights

• •Protocol to study mitochondrial structure, function, and metabolism comprehensively
• •Stepwise protocol for mitochondrial staining using confocal microscopy
• •Modular staining for multi-parameter assays
• •Mass spectrometry-based metabolomic analysis of mitochondrial metabolites

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

Mitochondria regulate a variety of biological activities, including metabolism, oxidative stress, and cell death. Here, we present a protocol for the investigation of mitochondrial structure, function, and metabolism in human cervical cancer cells. We describe steps for staining and visualizing mitochondria using confocal microscopy to assess morphology, mass, membrane potential, calcium, reactive oxygen species (ROS), and lipid droplet accumulation. We then detail procedures for isolating mitochondria and performing metabolomic analysis of mitochondrial metabolites via mass spectrometry.

Before you begin

The below step-by-step protocol explains the detailed procedure used to determine the mitochondrial structure, function and metabolism in cervical cancer cell lines. This protocol includes extensive cell culture techniques, and users should have specialized cell culture facilities, such as CO₂ 37°C incubators, cell culture hoods, microscopes and other related instruments, to perform below mentioned procedures. Furthermore, these techniques require confocal microscopy and mass spectrometry to carry out these experiments.

Innovation

In this protocol we have presented, a series of integrated, reproducible and high-resolution methods for the assessment of structure, function and metabolic integrity of the mitochondria in living cells. As compared to standard single -parameter analysis, our method allows visualization and quantification of several mitochondrial parameters simultaneously. The parameters such as mitochondrial mass, morphology, membrane potential, reactive oxygen species, Calcium, lipids and mitochondrial DNA integrity were present with established combinations of organelle-specific probes and confocal microscopy.

Our methodological innovation in the protocol includes a 48-h metabolic synchronization step that can reduce the basal metabolic noise and can enhance the sensitivity to mitochondrial perturbations. We have also included a modular staining strategy to preserve the integrity of living cells and simultaneous sequential imaging of distinct mitochondrial functions. We have meticulously standardized each assay for the concentration of fluorophore, incubation time and imaging depth to reduce variability across replicates and cell types. In addition, the use of Fiji-ImageJ-based quantitative workflows ensures objective, reproducible image analysis using automated thresholding, background subtraction, and ROI-based fluorescence quantification. This strategy bridges the imaging and biochemical accuracies making it more adaptable for the studies including drug-induced mitochondrial stress, mitochondria related cell death and toxic pathways. This method adapts to various cell models and is also compatible with mass spectrometry-based metabolomics. Further, this technique extends its potential on mitochondrial dysfunctions in metabolomic and cancer studies.

Institutional permissions

The cell lines used in this study were approved for research by the Manipal Academy of Higher Education. The study was approved by the Institution Research Committee, Manipal School of Life Sciences, Manipal and Biosafety Committee. Others who wish to replicate this protocol will need approval from their respective funding agencies and/or institutions.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium (DMEM), low glucose w/1 g glucose per litre, L-glutamine, sodium bicarbonate, and sodium pyruvate	HiMedia, India	AL006A
Fetal bovine serum	Gibco, Thermo Fisher Scientific Inc., USA	A5256701
Antibiotic Solution 100× Liquid, Endotoxin tested	HiMedia, India	A001A
Dulbecco’s modified Eagle’s medium (DMEM), low glucose w/1 g glucose per litre, L-glutamine, sodium bicarbonate, and sodium pyruvate w/o phenol red	HiMedia, India	AL183A
MitoTracker Red FM	Molecular Probes, USA	M22425
MitoSOX Red	Molecular Probes, USA	M36008
Rhodamine-123	Molecular Probes, USA	R302
Nonyl Acridine Orange	Thermo Fisher Scientific, USA	A1372
Pluronic F-127	Molecular Probes, USA	P3000MP
Rhod-2, AM, cell permeant	Molecular Probes, USA	R1244
Nile Red	Thermo Fisher Scientific, USA	N1142
Hoechst-33342	HiMedia, India	TC266
Pico Green	Thermo Fisher Scientific, USA	P7589
Methanol, Optima LC/MS grade	Thermo Fisher Scientific, USA	AAB-A456-4
Acetonitrile, LC-MS grade, 99.8%	Thermo Fisher Scientific, USA	AA47138K2
Formic acid, LC-MS grade	Thermo Fisher Scientific, USA	A117-50
Experimental models: Cell lines
SiHa (Human Cervical Squamous Cell Carcinoma)	ATCC	HTB-35
Software and algorithms
Leica Application Suite X (LAS X) software	Leica Microsystems, Germany	https://www.leica-microsystems.com
Fiji (ImageJ)	Schneider et al.2	https://imagej.net/Fiji
Other
Corning 75 cm2 U-shaped canted neck cell culture flask with vent cap	Corning, USA	430641U
Corning 150 mm TC-treated culture dish	Corning, USA	430599
Spinwin Tube conical bottom (15 mL tube)	Tarson, India	546041
35 mm glass bottom Petri plate	Ibidi, Germany	81218–200
8-well polymer coverslip	Ibidi, Germany	80806
Biological safety cabinets, Airstream Class II type A2, biological safety cabinet (S-series) NS	ESCO Lifesciences, Singapore	AC2-4S8-NS
Celculture@ CO2 incubator	ESCO Lifesciences, Singapore	CCL-170B-8
DMi8-TCS-SP8 confocal microscope	Leica Microsystems, Germany	Not applicable
Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer	Thermo Fisher Scientific, USA	Model: R-4C
Remi R-4C Centrifuge Machine	REMI Sales & Engineering Ltd., India	R-4C
Analytical C18 liquid chromatography (LC) column	Thermo Fisher Scientific, USA	Not applicable
Eppendorf Centrifuge 5415 R	Eppendorf AG, Hamburg, Germany	5415 R

Materials and equipment

Cell culture medium

Reagents	Final concentration	Amount
DMEM	1×	420 mL
FBS	10%	75 mL
Antibiotic Solution 100× Liquid, Endotoxin tested	100×	5 mL
Total	N/A	500 mL

• •The medium was filtered with a 0.22 μm filter unit.
• •Make several 200 mL aliquots in conical tubes to be used throughout the culture process, and label each with initial, date and time of preparation and expiration date.
• •The medium can be stored at 4°C for up to 4 weeks.

1× Phosphate buffer saline (pH 7.4)

Reagents	Final concentration	Amount
NaCl (Sodium chloride)	137 mM	8.00 g
KCl (Potassium Chloride)	2.7 mM	0.20 g
Na2HPO4 (Disodium phosphate, anhydrous)	10 mM	1.44 g
KH2PO4 (Monopotassium phosphate)	1.8 mM	0.24 g
Distilled water	N/A	1000 mL
Total	N/A	1000 mL

• •Add 800 mL of distilled water to all of the dry ingredients
• •Stir until all ingredients dissolved completely.
• •Use HCL or NaOH to bring the pH to 7.4, if necessary.
• •sterilize the Phosphate buffer saline by autoclaving and filtered with a 0.22 μm filter unit.
• •The Phosphate buffer saline can be stored at 4°C for up to 4 weeks.

Stock solution for assessment of mitochondrial morphology (MitoTracker Red FM)

• •Dissolve 50 μg of MitoTracker Red FM in 92 μL of DMSO to obtain a 1 mM stock solution.
• •Aliquot the reconstituted stock solution and store at −20°C for up to 6 months or −80°C for up to 1 year in the dark.

Alternatives: MitoTracker Green or Mito-BDP 630.

Stock solution for assessment of mitochondrial ROS (MitoSOX Red)

• •Dissolve 50 μg of MitoSOX Red in 13 μL of DMSO to obtain a 5 mM stock solution.
• •Aliquot and store the reconstituted stock solution at −20°C for up to 6 months or −80°C for up to 1 year away from light.

Alternatives: MitoBright ROS Deep Red, dihydroethidium (DHE), and probes such as mito-roGFP.

Stock solution for assessment of mitochondrial calcium

Rhod-2AM stock solution

• •Dissolve 50 μg of Rhod-2AM in 44.87 μL of DMSO to obtain a 1 mM stock solution.
• •Aliquot and store reconstituted stock solution at −20°C for up to 6 months or −80°C for up to 1 year in the dark.

Alternatives: Fluo-4AM (cytosol calcium (Ca²⁺)).

Hoechst-33342 stock solution

• •Dissolve 10 μg of Hoechst-33342in 10 mL of DMSO to obtain a 1 μg/mL stock solution.
• •Aliquot and store stock solution at 4°C to −20°C for up to 1 year in the dark.

Alternatives: DAPI.

Working solution for assessment of mitochondrial calcium

• •Add 0.01 mg Pluronic F-127 to 1 mL of HBSS buffer (1×) to obtain 0.01%.
• •Add 1 μL of Rhod2AM (1 mM stock) to above mixture to get 1 μM concentration.
• •Prepare fresh before use or store working solution at −20°C for up to 1 week.

Stock solution for assessment of mitochondrial mass (nonyl acridine orange)

• •Dissolve 1 mg of Nonyl Acridine Orange (NAO) in 211.64 μL of DMSO to obtain a 10 mM stock solution.
• •Aliquot and store reconstituted stock solution at −20°C for up to 6 months or −80°C for up to 1 year in the dark.

Stock solution for assessment of the mitochondrial membrane potential (rhodamine-123)

• •Dissolve 1 mg of Rhodamine-123 in 525 μL of DMSO to obtain a 5 mM stock solution.
• •Aliquot and store reconstituted stock solution at −20°C for up to 6 months or −80°C for up to 1 year in the dark.

Alternatives: MitoTracker Red, MitoTracker Orange, JC-1, TMRE (Tetra-methyl-rhodamine ethyl ester) and TMRM (Tetra-methyl-rhodamine methyl ester).

Stock solution for the localization of lipid droplets in mitochondria

Nile Red stock solution (5 μg/mL)

• •Dissolve 5 μg of Nile red in 1 mL of DMSO to obtain a 5 μg/mL stock solution.
• •Aliquot and store reconstituted stock solution at −20°C for up to 6 months or −80°C for up to 1 year in the dark.

Alternatives: BODIPY 493/503, LipidTOX Dyes, and Oil Red O.

Hoechst-33342 stock solution

• •Dissolve 10 μg of Hoechst-33342in 10 mL of DMSO to obtain a 1 μg/mL stock solution.
• •Aliquot and store stock solution at 4°C to −20°C for up to 1 year in the dark.

Alternatives: DAPI.

Rhodamine-123 stock solution (5 mM)

• •Dissolve 1 mg of Rhodamine-123 in 525 μL of DMSO to obtain a 5 mM stock solution.
• •Aliquot and store reconstituted stock solution at −20°C for up to 6 months or −80°C for up to 1 year in the dark.

Stock solution for analysis of mitochondrial DNA depletion

PicoGreen stock solution

• •Dissolve 1 μg of PicoGreen in 500 μL of DMSO to obtain a 1:500 solution.
• •The reconstituted stock solution was stored at −20°C for up to 6 months or −80°C for up to 1 year in the dark.

Alternatives: TOTO/YOYO dyes.

MitoTracker Red FM stock solution

• •Dissolve 50 μg of MitoTracker Red FM in 92 μL of DMSO to obtain a 1 mM stock solution.
• •Aliquot the reconstituted stock solution and store at −20°C for up to 6 months or −80°C for up to 1 year in the dark.

Alternatives: MitoTracker Green or Mito-BDP 630.

Stock solution for metabolomic analysis of mitochondrial metabolites via mass spectrometry

Reagents	Final concentration	Amount	Notes
Methanol	50 %	5 mL	LC-MS Grade ≥99.9 %
Acetonitrile	30 %	3 mL	LC-MS Grade ≥99.9 %
Water	20 %	2 mL	LC–MS grade/Milli-Q
Total	100 %	10 mL

• •Mix all 3 components in pre-chilled 15 mL tube.

5 mL of Methanol.

3 mL Acetonitrile.

2ml of Water.

Mix thoroughly by vertexing or gentle inversion.

• •Use immediately or store the mixture at −80°C for long-term use or,

Step-by-step method details

Cell culture: Culturing and maintaining the cell line

Timing: 3–4 days

The protocol below explains the steps for culturing and maintaining human cervical cancer cell lines (SiHa cells) obtained from the American Type Culture Collection (USA).

Note: We used cervical cancer cell lines for this protocol, and users can replicate them in any cell line.

• 1.
  Culturing SiHa cells.

  • a.
    Prewarm Dulbecco’s modified Eagle’s medium (DMEM), low glucose weight/1 gram glucose per litre, L-glutamine, sodium bicarbonate and sodium pyruvate supplemented with 10% fetal bovine serum (complete medium) and 1× phosphate-buffered saline in a 37°C water bath for up to 30 min.
  • b.
    Bring the complete medium into a laminar flow hood.
  • c.
    Next, trypsinize the SiHa cells from routine passage and seed 1 × 10⁶ SiHa cells to a 75 cm² culture flask in a laminar flow hood.
  • d.
    Maintain and culture the cells in 15 mL of DMEM supplemented with 10% fetal bovine serum (complete media).
  • e.
    Incubate the cells at 37°C with 5% CO₂.
  • f.
    Once the cells reached 80–90% confluence, the medium was removed from the flask, and the cells are washed twice with 2 mL of 1× phosphate-buffered saline (pre warmed).
  • g.
    After washing, the cells are treated with 1 mL of 0.5% trypsin-EDTA solution and incubated at room temperature (25°C–30°C) for 30 s.
  • h.
    The trypsin-EDTA solution is aspirated, and the flask is incubated at 37°C with 5% CO₂ for 2 min.
  • i.
    After incubation, the flask is tapped gently for complete detachment of the cells from the surface of the flask.
  • j.
    Check under the microscope if the cells have detached from the plate.

    CRITICAL: Ensuring that all cells have detached before proceeding to the next step is important (Figure 1).

  • k.
    Once the cells detached from the flask surface, 4 mL of complete media was added to block trypsin activity.
  • l.
    Transfer the cell suspension to a 15 mL tube and centrifuge at 300 × g for 5 min at room temperature (25°C–30°C).
  • m.
    After centrifugation, Discard the supernatant. Pellets are resuspended in fresh complete medium for further studies.

Figure 1.

Morphological changes of SiHa cells before and after trypsinization

(A) Phase contrast image showing elongated polygonal shaped cells with homogenous monolayer distribution.

(B) Phase contrast image showing round and detached cells after treatment with 0.5% trypsin-EDTA solution (magnification 10×).

Mitochondrial morphology assessment via MitoTracker Red

Timing: ∼3 days 1 h

Below is the step-by-step protocol used to determine mitochondrial morphology in SiHa cells.

• 2.
  Preparing the cells for Mito Tracker Red staining.

  • a.
    Seed SiHa cells in 35 mm glass bottom Petri plates, at a concentration of 3 × 10⁴ cells/plate.
  • b.
    Add 1 mL of complete media to the plate.
  • c.
    Incubate plate at 37°C with 5% CO_2.
  • d.
    Aspirate the complete media once the cells reach the 70 % confluency.

    CRITICAL: Make sure the cells are well attached to the surface of the plate.

  • e.
    Wash the cells twice with 1 mL of PBS (5 min each).
  • f.
    Cells in the petri plates are then starved for 48 h in 1 mL of serum-free DMEM media (SFM) at 37°C with 5% CO_2.

    CRITICAL: In this protocol, we have starved cells for 48 h to achieve high degree of metabolic synchronization and supress the baseline inhibition of growth signaling. However, prolonged starvation can induce autophagy and other stress related metabolic reprograming that may hamper the mitochondria structure and function. We advise researcher working with drug responses, and signaling pathways should standardize serum deprivation periods based on their experimental goals. Troubleshooting 1.

  • g.
    After 48 h of starvation, wash the plates twice with 1 mL of PBS (5 min each).
• 3.
  Mito-Tracker Red staining.

  • a.
    Stain the cells with 1 μL of 1 mM MitoTracker Red (Ex/Em: ∼581/644 nm) in 1 mL of SFM.

    CRITICAL: Protect the dye from light and avoid repeated freeze thaw cycles. Troubleshooting 2.

  • b.
    Incubated at 37°C with 5% CO₂ for 30 min in the dark.

    Note: Less or more incubation time can lead to inappropriate staining; we recommend that users standardize the incubation time for each cell line before starting the experiments. Troubleshooting 3.

  • c.
    After incubation, wash the plates thrice with 1 mL of PBS (5 min each) to remove excessive stains.

    Note: Proper washing is necessary to remove excessive stain.

  • d.
    Add 1 mL of phenol red-free SFM to the plate.
• 4.
  Visualization of stained cells

  • a.
    Observe stained cells under a DMi8-TCS-SP8 confocal microscope with a 63× oil immersion objective.
  • b.
    Images were taken in point-scanning mode as Z-stacks with an optimum step size, covering a total depth of 2432.64 μm, with the pinhole set at 95.5 μm (1 Airy unit).

CRITICAL: Avoid direct contact of stained cells with light.

• 5.Process image using LAS X software.
  Step by step protocol for image acquisition and processing using Las-X software (Figure 2).

  • a.
    Launch the LAS X software and click on “Open project”.
  • b.
    Then, select required acquisition mode and objective setting.
  • c.
    Place prepared stained cells onto the microscope stage.
  • d.
    Navigate to the “acquire” tab and tweak channel-specific parameters like the laser line, gain, and offset.
  • e.
    Use the “Laser Settings” panel to turn on and adjust the laser intensities for each fluorescence channel.
  • f.
    Live view of the cells is displayed in the lower panel with real-time channel visualization.
  • g.
    After capturing the desired image, process the image by navigate the “process” tab in the main menu.
  • h.
    Edit the image for cropping, merging, filtering and contrast enhancement using image processing option in the software.
  • i.
    Choose the right save and export settings to export the finished processed images in TIFF format.

Figure 2.

Workflow for image acquisition and processing using Leica LAS X software

Mitochondrial ROS production via MitoSOX Red

Timing: ∼3 days 45 min

Below is the step-by-step protocol used to determine mitochondrial ROS production in SiHa cells.

• 6.
  Preparing cells for MitoSOX Red staining.

  • a.
    Culture SiHa cells (3 × 10⁴ cells/plate) in 35 mm glass bottom Petri plates and starved for 48 h in SFM as described in mitochondrial morphology assessment via MitoTracker Red.

    CRITICAL: Make sure the cells are well attached to the surface of the plate.

  • b.
    After 48 h of starvation, plates were washed twice with 1 mL of PBS (5 min each).
• 7.
  Mito SOX red staining.

  • a.
    Stain the cells with 0.1 μL of 5 mM MitoSOX red (Ex/Em: ∼396/610 nm) in 1 mL of SFM.
  • b.
    Incubate at 37°C with 5% CO₂ for 15 min in the dark.

    Note: Less or more incubation time can lead to inappropriate staining, and we recommend that users standardize the incubation timing for each cell line before starting the experiment. Troubleshooting 3.

  • c.
    After incubation, wash the plates twice with 1 mL of PBS (5 min each) to remove excess stain.

    CRITICAL: Proper washing is necessary to remove excessive stain.

  • d.
    Add 1 mL of phenol red-free SFM was added to the plate.
• 8.
  Visualization and analysis of stained cells.

  • a.
    The stained cells were observed under a Leica DMi8 TCS SP8 confocal microscope with a 63× oil immersion objective.

    Note: Images were taken in point-scanning mode as z stacks with an optimum step size, covering a total depth of 1489.9 μm, with the pinhole set at 95.5 μm (1 Airy unit).

    CRITICAL: Direct exposure of stained cells to light should be avoided.

  • b.
    Quantify mitochondrial ROS by measuring the mean fluorescence intensity of cells from random fields via LASX software as explained in step 5 and quantify mitochondrial ROS using Fiji-ImageJ software.
• 9.Quantification of mitochondrial ROS using Fiji-ImageJ software.
  Step by step protocol for image quantification using Fiji-ImageJ.

  • a.
    Use File → Open in Fiji/ImageJ to open the exported TIFF files.
  • b.
    To measure intensity accurately, convert each image to 32-bit format: Image → Type → 32-bit.
  • c.
    Use the rolling ball technique with a 50-pixel radius to eliminate background fluorescence: Process → Subtract Background → Rolling ball radius: 50 → OK.
  • d.
    Use Otsu’s automated technique to segment cells by applying an intensity threshold: Image → Adjust → Threshold → Select “Otsu” method → Apply.
  • e.
    Use the thresholder image’s particle detection to identify cells: Analyze → Analyze Particles.
  • f.
    Set the circularity range to 0.5–1.0 and the size range to 50–1000 μm².
  • g.
    To save ROIs, select “Add to Manager”.
  • h.
    Measure the fluorescence intensity of each ROI after selecting them all in the ROI Manager: Analyze → Measure.
    For every ROI, note the average intensity values.
  • i.
    Choose three cell-free regions as background ROIs from each image manually, then calculate the mean fluorescence intensity of these regions.
  • j.
    Determine the mean fluorescence intensity of each cell ROI by subtracting the average background intensity from the three background ROIs.
  • k.
    Save the measurement data as an Excel or CSV file for later statistical examination.

Mitochondrial calcium production via Rhod2AM

Timing: ∼3 days 30 min

Below is the step-by-step protocol used to evaluate mitochondrial calcium (mtCa²⁺) levels via Rhod2AM.

• 10.
  Preparing cells for Rhod2AM staining.

  • a.
    Culture SiHa cells (3 × 10⁴ cells) were cultured in 8-well polymer coverslips (Ibidi, Germany) containing 300 μL of complete media.
  • b.
    Incubate cells at 37°C with 5% CO₂.
  • c.
    Once the cells reach 70% confluency remove, the complete media.

    CRITICAL: Ensure that the cells are well attached to the surface of the plate.

  • d.
    The cells were washed twice with 300 μL of HBSS buffer (5 min each).
  • e.
    Starved the cells in 300 μL of SFM at 37°C with 5% CO₂ for 48 h.
  • f.
    After 48 h of starvation, wash plates twice with 300 μL of HBSS buffer (5 min each).
• 11.
  Rhod2AM staining.

  • a.
    Stain the cells with 0.5 μL of 1 μM Rhod2AM (Ex/Em: ∼ 552/581 nm) and 1 μL of Hoechst 33342 (Ex/Em: ∼ 361/486 nm) dyes to stain mtCa²⁺ and the nucleus, respectively.
  • b.
    Incubate the cells in the dark at 37°C with 5% CO₂ for 30 min.

    CRITICAL: Less or more incubation time can lead to inappropriate staining, and we recommend that users standardize the incubation timing for each cell line before starting the experiment. Troubleshooting 3.

  • c.
    After staining, the cells were washed three times with 300 μL of HBSS (5 min each) to remove excess stain.

    CRITICAL: Proper washing is necessary to remove excessive stain.

  • d.
    Add 1 mL of phenol red-free SFM to the plate.
• 12.
  Visualization of stained cells.

  • a.
    The live imaging was performed via a Leica DMi8 TCS SP8 confocal microscope with a 63× oil immersion objective.

    Note: Images were taken in point-scanning mode as z stacks with an optimum step size, covering a total depth of 2384.43 μm, with the pinhole set at 95.5 μm (1 Airy unit).

    CRITICAL: Direct exposure of stained cells to light should be avoided.

  • b.
    Process the image using LAS-X software as explained in step 5 and quantify the images using Fiji-ImageJ as detailed in step 9.

Mitochondrial mass via nonyl acridine orange

Timing: ∼3 days 1 h

Below is the step-by-step protocol used to evaluate mitochondrial mass via the Nonyl Acridine Orange.

• 13.
  Preparing cells for nonyl acridine orange staining.

  • a.
    Seed SiHa cells (3 × 10⁴ cells) in 8-well polymer coverslips (Ibidi, Germany) and starved for 48 h in SFM as described in step 10.

    CRITICAL: Ensure that the cells are well attached to the surface of the plate.

  • b.
    After starvation, wash the cells twice with 300 μL of PBS (5 min each).
• 14.
  Nonyl acridine orange staining.

  • a.
    MM was evaluated by incubating the cells with staining solution containing 2 μL of Nonyl Acridine Orange (Ex/Em: ∼496/519 nm).

    CRITICAL: Protect the dye from light and avoid repeated freeze thaw cycles. Troubleshooting 2.

  • b.
    Incubate cells for 30 min in the dark at 37°C.

    CRITICAL: Less or more incubation time can lead to inappropriate staining; we recommend that users standardize the incubation time for each cell line before starting the experiments. Troubleshooting 3.

  • c.
    Excess stains were removed by washing with 300 μL of PBS (twice, 5 min each).

    CRITICAL: Proper washing is necessary to remove excessive stain.

  • d.
    Add 300 μL of phenol red-free SFM to the coverslip.
• 15.
  Visualization and analysis of stained cells.

  • a.
    The stained cells were imaged via a Leica DMi8 TCS SP8 confocal microscope with a 63× oil immersion objective.

    Note: Images were taken in point-scanning mode as z stacks with an optimum step size, covering a total depth of 2432.64 μm, with the pinhole set at 95.5 μm (1 Airy unit).

    CRITICAL: Direct exposure of stained cells to light should be avoided.

  • b.
    Process the image using LAS-X software as explained in step 5 and quantify the images using Fiji-ImageJ as detailed in step 9.

Mitochondrial membrane potential determination via rhodamine-123

Timing: 3 days 1 h

Below is the stepwise protocol used to measure the mitochondrial membrane potential via Rhodamine 123.

• 16.
  Preparing cells for Rhodamine 123 staining.

  • a.
    Seed SiHa cells (3 × 10⁴ cells) in 8-well polymer coverslips (Ibidi, Germany) and starved for 48 h in SFM as described in step 10.

CRITICAL: Ensure that the cells are well attached to the surface of the plate. After starvation, wash the cells twice with 300 μL of PBS (5 min each).

• 17.
  Rhodamine 123 staining.

  • a.
    The extent of the MMP was quantified by incubating the cells with staining solution containing 2 μL of 5 mM Rhodamine-123 (Ex/Em: ∼508/528 nm).

    CRITICAL: Protect the dye from light and avoid repeated freeze thaw cycles trouble shooting Troubleshooting 2.

  • b.
    Incubate the cells for 30 min in the dark at 37°C.

    CRITICAL: Less or more incubation time can lead to inappropriate staining; we recommend that users standardize the incubation time for each cell line before starting the experiments. Troubleshooting 3.

  • c.
    Excess stains were removed by washing with 300 μL of PBS (twice, 5 min each).

    CRITICAL: Proper washing is necessary to remove excessive stain.

  • d.
    Add 300 μL of phenol red-free SFM to the coverslip.
• 18.
  Visualization and analysis of stained cells.

  • a.
    The stained cells were imaged via a Leica DMi8 TCS SP8 confocal microscope with a 63× oil immersion objective.

    Note: Images were taken in point-scanning mode as z stacks with an optimum step size, covering a total depth of 2479.93 μm, with the pinhole set at 95.5 μm (1 Airy unit).

    CRITICAL: Direct exposure of stained cells to light should be avoided.

  • b.
    Process the image using LAS-X software as explained in step 5 and quantify the images using Fiji-ImageJ as detailed in step 9.

Localization of lipid droplets in mitochondria

Timing: 3 days 1 h 30 min

Below is the stepwise protocol to localize lipid droplets in mitochondria via Nonyl acridine orange and Rhodamine 123.

• 19.
  Preparing cells for staining.

  • a.
    Seed SiHa cells (3 × 10⁴ cells) 8-well polymer coverslips (Ibidi, Germany) and starved for 48 h in SFM as described in step 10.

    CRITICAL: Ensure that the cells are well attached to the surface of the plate.

  • b.
    After starvation, wash the cells twice with 300 μL of PBS (5 min each).
• 20.
  Nonyl acridine orange staining.

  • a.
    Incubate the cells with staining solution containing 2 μL of Rhodamine-123 (Ex/Em: ∼508/528 nm) for 30 min in the dark at 37°C.
  • b.
    Excess stain was removed by washing with 300 μL of PBS (twice, 5 min each).
  • c.
    After washing, co-stain the cells with 0.1 μL of Nile Red (Ex/Em: ∼552/636 nm) and 1 μL of Hoechst 33342 (Ex/Em: ∼361/486 nm).
  • d.
    Incubate cells for 30 min in the dark at 37°C.

    CRITICAL: Less or more incubation time can lead to inappropriate staining; we recommend that users standardize the incubation time for each cell line before starting the experiments. Troubleshooting 3.

  • e.
    Excess stain was removed by washing with 300 μL of PBS (twice, 5 min each).

    CRITICAL: Proper washing is necessary to remove excessive stain.

  • f.
    Add 300 μL of phenol red-free SFM to the coverslip.
• 21.
  Visualization and analysis of stained cells.

  • a.
    The stained cells were imaged via a Leica DMi8 TCS SP8 confocal microscope with a 63× oil immersion objective.

    Note: Images were taken in point-scanning mode as z stacks with an optimum step size, covering a total depth of 2450.63 μm, with the pinhole set at 95.5 μm (1 Airy unit).

    CRITICAL: Direct exposure of stained cells to light should be avoided.

  • b.
    Process the image using Image processing via LAS-X software as described in section in step 5.
  • c.
    Quantify the images using Fiji-ImageJ software as detailed in step 9.

Analysis of mitochondrial DNA depletion content via PicoGreen dye

Timing: 3 days 30 min

Below, a step-by-step protocol was used to visualize the depletion of mtDNA content in SiHa cells via PicoGreen stain.

• 22.
  Preparing cells for staining.

  • a.
    SiHa cells (3 × 10⁴ cells) were seeded on 8-well polymer coverslips (Ibidi, Germany) and starved for 48 h in SFM as described in step 10.

    CRITICAL: Ensure that the cells are well attached to the surface of the plate.

  • b.
    After starvation, the cells were washed twice with 300 μL of PBS (5 min each).
• 23.
  Co-staining with PicoGreen and MitoTracker Red.

  • a.
    After washing, stain the cells with 1 μL PicoGreen (Ex/Em: ∼ 361/486 nm) and 1 μL of MitoTracker Red (Ex/Em: ∼ 581/644 nm).
  • b.
    Incubate cells for 30 min at 37°C in the dark.

    CRITICAL: Less or more incubation time can lead to inappropriate staining; we recommend that the authors standardize the incubation time for each cell line before starting the experiments. Troubleshooting 3.

  • c.
    After incubation, wash the cells twice with 300 μL of PBS (5 min each).

    CRITICAL: Proper washing is necessary to remove excessive stain.

  • d.
    Add 300 μL of colorless, serum-free DMEM to an 8-well polymer coverslip.
• 24.
  Visualization and analysis of stained cells.

  • a.
    The stained cells were imaged using a Leica DMi8 TCS SP8 confocal microscope with a 63× oil immersion objective Images were taken in point-scanning mode as Z-stacks with an optimum step size, covering a total depth of 4469.7 μm, with the pinhole set at 95.5 μm (1 Airy unit).

    CRITICAL: Direct exposure of stained cells to light should be avoided.

  • b.
    To identify the mitochondrial DNA, green and red signals were merged via LAS-X software as described in step 5.
  • c.
    Quantify the images via Fiji-ImageJ software as detailed in step 9.

Metabolomic analysis of mitochondrial metabolites via mass spectrometry

Timing: 4 days 5 h

Below is the step-by-step procedure for sample preparation and metabolomic analysis of mitochondrial metabolites via mass spectrometry.

• 25.
  Sample preparation for mass spectrometry.

  • a.
    Seed SiHa cells (3 × 10⁵ cells) in 10 cm plates and incubated for 24 h under standard conditions for cell attachment.

    Note: Make sure the cells are well `attached to the surface of the plate

  • b.
    The cells were then subjected to 48 h of serum starvation.
  • c.
    After starvation, collect around 1 million cells using trypsinization.

    Note: Based on sample requirements and assay sensitivity, cell number may be adjusted.

  • d.
    Centrifuge the cells at 100 × g for 5 min to collect the pellets.
  • e.
    Wash the pellets twice with 1 mL of PBS.
• 26.
  Isolation of mitochondria from trypsinized cells.

  • a.
    Isolate mitochondria from trypsinized cells using method published by Spinazzi et al.³
  • b.
    After trypsinization and PBS Wash, discard the supernatant and flash freeze the cells in 15 mL tube.
  • c.
    Thaw the cell pellets at 37°C and add 1 mL of ice -cold 10 mM hypotonic tris buffer (pH 7.6).
  • d.
    homogenizes the cell mixture using 2-ml glass/Teflon tissue grinder with a tight clearance kept on ice with 15 slow up-down strokes at 1800 rpm.

    Note: Before beginning the homogenization process, let the tissue grinder cool on ice for 5 min. To prevent microbiological and chemical contamination, tissue grinders need to be thoroughly cleaned and dried before analysis.

  • e.
    To the cell homogenate, add 200 μL of a 1.5 M sucrose solution and stir well.
  • f.
    After sucrose addition, centrifuge the cell mixture at 600 × g for 10 min 2°C.
    After collecting the supernatant, transfer it to a 1.5 ml microcentrifuge tube and centrifuge it for 10 min at 2°C at 14,000 × g.
  • g.
    Carefully discard the supernatant. add 200 μL of a pre-chilled methanol: acetonitrile: water solution mixture (5:3:2) to the washed mitochondria pellets, vortex or pipette up and down the mixture and store at in −80 12 h for the cell lysis and metabolite extraction.
  • h.
    After incubation, centrifuge the sample at 20,000 × g for 20 min at 4°C to separate the protein.
  • i.
    Collect the supernatant and concentrate using a vacuum concentrator to set the V-AQ mode at 30°C for 4 h.
  • j.
    Add 30 μL of 50% acetonitrile and 0.1% formic acid mixture to the concentrated sample, vortex for 5 min.
  • k.
    After vortex, centrifuge the sample at 13000 × g for 10 min.
  • l.
    After centrifugation, transfer the 30 μL of content to HPLC tubes for LC/MS analysis.
• 27.
  Instrument set up and sample analysis.

  • a.
    All samples will be run in triplicate via the Q Exactive Plus (Thermo Scientific) in electrospray ionization (ESI) positive/negative mode.
  • b.
    Solvent (A), consisting of water with 0.1% formic acid, and solvent (B), consisting of methanol with 0.1% formic acid, will be used for a 35-min run on an analytical C18 liquid chromatography (LC) column with dimensions of 250 mm × 4.6 mm at 0.5 ml/min flow rates will be specified per the method.
  • c.
    Parameters such as capillary temperature (320°C), sheath gas flow rate (60 AU), auxiliary gas flow rate (20 AU), and spray voltage (3.5 kV) set to default values. The instrument will be operated via Thermo Scientific Xcalibur software.
• 28.
  Data analysis.

  • a.
    Analyze the data using Human Metabolome Database (HMDB; www.hmdb.ca/) and MetaboAnalyst-5.0 (www.metaboanalyst.ca/) databases for metabolomics data analysis.⁴
  • b.
    To assign metabolite IDs, raw MS peaks were curated and annotated against the HMDBMetaboAnalyst 5.0 (www.metaboanalyst.ca/) was updated with annotated metabolites along with their intensity values.
  • c.
    Before analysis, the data were Pareto-scaled and log-transformed.
  • d.
    The hypergeometric test was used for pathway enrichment, and relative-betweenness centrality was used to assess pathway structure.
  • e.
    Organism was set to Homo sapiens.
  • f.
    The FDR-adjusted p-value, impact score, and enrichment ratio were used to rank the pathways.
  • g.
    The results were displayed using pathway tables and bubble plots in accordance with Adiga et al.¹ methodology.

Expected outcomes

In this protocol, we aim to investigate how genetically modified DOC2B gene can effectively modulate mitochondrial structure, function and metabolism in cervical cancer cells. In our previous study, we developed DOC2B overexpression and knockdown model systems in cervical cancer cells. The overexpression of DOC2B revealed that DOC2B is in mitochondria and causes Ca²⁺-mediated lipotoxicity.¹ High resolution Confocal microscopy with mitochondria specific dye such as Mito Tracker red demonstrated altered mitochondria morphology in DOC2B modified cells (Figure 3). Overexpression or knockdown of DOC2B showed fragmented or condensed mitochondrial network, decreased membrane potential and mitochondrial mass using Rhodamine 123 and Nonyl acridine orange (Figure 4). DOC2B overexpression also increased mitochondrial Ca²⁺ and mitochondria ROS in SiHa cells, which is indicated by increased Rhod2AM and MitoSOX Red signal intensity in mitochondria, respectively (Figure 5). Nile red depicts the lipid droplet accumulation in and around mitochondria, counterstaining with Rhodamine 123 locate lipid droplet in mitochondria. Altered Nile red intensity indicates that DOC2B overexpression resulted in increased lipid droplet accumulation inside the mitochondria. Picogreen dye is an indicator of DNA content depletion in the cell, counter staining with mito tracker red revealed that DOC2B overexpression modulated DNA content in mitochondria (Figure 6). In response to DOC2B overexpression, mitochondrial metabolomics data revealed considerable enrichment of metabolites associated with arachidonic acid metabolism, phospholipid biosynthesis, mitochondrial β-oxidation of long chain saturated FAs, fatty acid (FA) biosynthesis, and FA metabolism (Figure 7).

Figure 3.

Representative results obtained from confocal microscopy analysis of mitochondrial morphology via MitoTracker Red

Published with permission from Elsevier. Sample results obtained via confocal microscope analysis are shown in Adiga et al.¹

Figure 4.

Representative results obtained from confocal microscopy analysis of mitochondria mass and mitochondrial membrane potential

Sample results obtained via confocal microscope analysis are shown in Adiga et al.¹

(A) Mitochondrial mass (MM) via nonyl acridine orange.

(B) Mitochondrial membrane potential (MMP) via Rhodamine 123. The data are presented as the means ± SDs from an independent set of experiments. The groups were compared using a two-tailed Student’s t-test. ∗∗ indicates p < 0.05, which was considered statistically significant.Published with permission from Elsevier.

Figure 5.

Representative results obtained from confocal microscopy analysis of mitochondrial calcium (mtCa2+) and mitochondrial ROS

Sample results obtained via confocal microscope analysis are shown in Adiga et al.¹

(A) Mitochondrial calcium (mtCa2+) via Rhod2AM.

(B) Mitocondrial ROS via MitoSOX Red. The data are presented as the means ± SDs from an independent set of experiments. The groups were compared using a two-tailed Student’s t-test. ∗∗ indicates p < 0.05, which was considered statistically significant. Published with permission from Elsevier.

Figure 6.

Representative results obtained via confocal microscopy showing mitochondrial lipid accumulation and mitochondrial DNA depletion

The sample results obtained are shown in Adiga et al.¹

(A) Confocal images of Rhodamine-123 and Nile red dual staining to confirm the mitochondrial localization of lipid droplets in DOC2B-manipulated cell lines. Colocalization analysis was performed via LASX software.

(B) Confocal images and bar graph showing DNA depletion images obtained via PicoGreen and MitoTracker Red in DOC2B manipulated cell lines. The groups were compared using a two-tailed Student’s t-test. ∗∗ indicates p < 0.05, which was considered statistically significant.

Figure 7.

Representative results obtained from the metabolomic analysis of mitochondrial metabolites via mass spectrometry

(A–D) The bubble plots represent the metabolite set enrichment in DOC2B overexpressing SiHa and vector-transfected cells. The sample results obtained via mass spectrometry are shown in Adiga et al.¹ Published with permission from Elsevier. The bubble plots represent the metabolite set enrichment in DOC2B-overexpressing SiHa and vector-transfected cells. The x-axis indicates the enrichment ratio, while the bubble size reflects the relative contribution of each pathway. The bubble color corresponds to the statistical significance (p-value), where values closer to 1.0 indicate higher statistical confidence of pathway enrichment, and lower values indicate weaker enrichment.

Limitations

In this study, we investigated different mitochondrial parameters via confocal microscopy and mass spectrometry in cervical cell lines. Therefore, to execute the same protocol with other cell lines, researchers need to standardize and optimize the experimental conditions, fluorescent dye concentration, incubation time and microscope setting for their own cell lines.

Troubleshooting

Problem 1

Serum starving the cells for 48 h can lead to altered drug response and mitochondria structure and function.

Potential solution

Standardize the serum starvation timings based on assay requirements. Monitor the autophagy and stress related marker to make sure cells are healthy.

Problem 2

Repeated freezing and thawing cycles of fluorescence dyes can lead to reduced fluorescence intensity in cell lines.

Potential solution

This is due mainly to factors such as denaturation, aggregation, and the formation of ice crystals that damage the sample, leading to dye degradation and loss of fluorescence. Small aliquots of fluorescence mixture were prepared and stored at the desired temperature.

Problem 3

Longer or shorter incubation times for fluorescence dyes can lead to inappropriate staining.

Potential solution

Nonspecific, weak, excessively intense, or uneven staining patterns that do not correctly represent mitochondrial morphology, localization, or abundance are referred to as inappropriate staining. This is mainly due to the amount of dye internalized by the cell line. While longer incubation can lead to non-specific staining or cause cell toxicity, whereas shorter incubation can result in weaker signal. We strongly recommend user should standardize the dye concentration and incubation time before conducting an actual experiment. Users can also check under a fluorescence microscope once before removing the dye from the cells.

Problem 4

Selecting the wrong chamber slides, coverslips or Petri dishes can lead to inappropriate staining and decreased image quality.

Potential solution

Chamber slides, coverslips or Petri dishes compatible with confocal microscopy were selected. This increases the image quality.

Problem 5

Diffuse/non-mitochondrial localization may be observed when staining for mitochondrial mass (NAO) or mitochondrial calcium (Rhod-2 AM).

Potential causes

• •Damaged mitochondria or increased NAO concentration.
• •Inadequate Rhod-2 AM de-esterification.
• •Excessive cytosolic dye load.
• •Low esterase activity.
• •Loss of mitochondrial membrane potential (Δψm).

Potential solution

• •Reduce non-specific signals by using a lower concentration of NAO.
• •For staining, make sure the mitochondria are robust and healthy.
• •Before imaging, provide enough time for Rhod-2 AM de-esterification.
• •Optimize dye loading to minimize cytosolic accumulation.
• •Prevent extended incubation or stressful situations to maintain Δψm.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shama Prasada K (shama.prasada@ibp.ac.cn).

Technical contact

For technical specifics on executing the protocol, Dr. Sachin Shetty (sachinshetty304@gmail.com) will provide support to ensure its correct implementation.

Materials availability

This study did not generate new unique reagents and materials.

Data and code availability

Original/source data for the figures presented in this paper are available at https://doi.org/10.1016/j.freeradbiomed.2023.03.010.

Acknowledgments

We would like to thank all members of the Department of Cell and Molecular Biology, Manipal School of Life Sciences, MAHE Manipal, for discussion. This work was supported by DBT, Government of India, under the DBT Wellcome Trust India Alliance (IA), India grant (grant no. IA/I/22/1/506240)).

Author contributions

Conceptualization and writing – original draft, S.S.; writing – review and editing, S.S. and S.P.K.; funding acquisition and supervision, S.P.K.

Declaration of interests

The authors declare no competing interests.