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Guide to the Seahorse Assay for Mitochondrial Respiration (Mito Stress Test)
- Mar 27
- 5 min read
In the field of cellular bioenergetics, the Agilent Seahorse XF Cell Mito Stress Test stands as the gold standard for quantifying mitochondrial function. Unlike end-point assays that provide a static snapshot of metabolism (like ATP lysis kits), the Seahorse assay measures the kinetics of mitochondrial respiration in real-time using live cells.
This comprehensive guide synthesizes data from peer-reviewed literature to provide a robust, step-by-step protocol for measuring Oxygen Consumption Rate (OCR) and determining the key parameters of mitochondrial health.
Did you know you can get a customized version of this protocol from Soφ AI? You might also be interested in this MTT vs L/D staining protocol!
Principle of the Assay: How It Works
The Seahorse XF Analyzer utilizes a transient microchamber within a specific culture plate (24-well or 96-well). Sensor cartridges, embedded with solid-state optical sensors, lower into the wells to create a temporary sealed chamber (typically 2–4 µL volume) above the cell monolayer.
Oxygen Consumption Rate (OCR): The sensors measure the decrease in O₂ concentration over time, which correlates to mitochondrial respiration (oxidative phosphorylation).
Extracellular Acidification Rate (ECAR): Simultaneously, the sensors measure the increase in protons (H⁺), primarily derived from glycolysis (lactate production).
The "Stress" in Mito Stress Test
The power of this assay lies in the sequential injection of four pharmacological modulators. By inhibiting specific complexes of the Electron Transport Chain (ETC), the assay dissects the total OCR into distinct functional components.
Injection | Compound | Target | Effect |
Basal | None | N/A | Measures baseline respiration under normal conditions. |
Port A | Oligomycin | Complex V (ATP Synthase) | Inhibits ATP synthesis. The drop in OCR represents oxygen used for ATP production. |
Port B | FCCP | Uncoupler (Proton Ionophore) | Collapses the proton gradient, forcing the mitochondria to work at maximum capacity to restore potential. |
Port C | Rotenone + Antimycin A | Complex I & III | Shuts down the ETC completely. Remaining OCR is non-mitochondrial. |
Key Bioenergetic Parameters Explained
Correctly interpreting Seahorse data requires understanding exactly what each phase of the OCR profile represents.
1. Basal Respiration
Definition: The energetic demand of the cell under baseline conditions.
Calculation: (Last rate before Oligomycin) - (Non-Mitochondrial Respiration)
2. ATP-Linked Respiration (ATP Production)
Definition: The portion of basal respiration used to drive ATP synthesis.
Calculation: (Last rate before Oligomycin) - (Minimum rate after Oligomycin)
3. Proton Leak
Definition: Basal respiration not coupled to ATP synthesis. This often indicates mitochondrial damage or uncoupling protein activity.
Calculation: (Minimum rate after Oligomycin) - (Non-Mitochondrial Respiration)
4. Maximal Respiration
Definition: The maximum rate of respiration the cell can achieve when physiologically stressed.
Calculation: (Maximum rate after FCCP) - (Non-Mitochondrial Respiration)
5. Spare Respiratory Capacity
Definition: The cell's ability to respond to an energetic demand. Higher capacity indicates better fitness and flexibility.
Calculation: (Maximal Respiration) - (Basal Respiration)
6. Non-Mitochondrial Respiration
Definition: Oxygen consumption due to non-mitochondrial oxidases and enzymes.
Calculation: Minimum rate after Rotenone/Antimycin A
Step-by-Step Protocol
This protocol merges the standard Agilent guidelines with specific optimizations for T cells and adherent cell lines found in STAR Protocols.
Phase 1: Preparation (Day -1)
A. Sensor Cartridge Hydration
Critical Step: The sensor cartridge must be hydrated overnight to ensure accurate pH and O₂ readings.
Pipette 200 µL of XF Calibrant into each well of the Utility Plate (96-well format) or 500 µL (24-well format).
Lower the Sensor Cartridge onto the Utility Plate. Ensure the sensors are submerged.
Incubate overnight at 37°C in a non-CO₂ incubator.
Note: Do not use a CO₂ incubator, as CO₂ absorption can skew the calibration of the pH sensors.
B. Cell Seeding (Adherent Cells)
Optimization: Determine optimal seeding density (typically 10k–80k cells/well for 96-well). A monolayer of 80–90% confluence is ideal.
Seed cells in standard culture medium (e.g., DMEM + 10% FBS).
Leave 4 corner wells (A1, A12, H1, H12) empty (medium only) for Background Correction.
Allow cells to adhere overnight in a standard cell culture incubator (37°C, 5% CO₂).
Phase 2: Assay Setup (Day 0)
A. Assay Medium Preparation
Seahorse requires specific "bicarbonate-free" medium to allow for accurate pH sensing.
Base Medium: Use Seahorse XF DMEM or RPMI (depending on cell type).
Supplements: Add substrates relevant to your cell type. Standard concentrations are:
Glucose: 10 mM
Pyruvate: 1 mM
Glutamine: 2 mM
pH Adjustment: Warm medium to 37°C and adjust pH to 7.4 using 1N NaOH. Filter sterilize (0.22 µm).
Warning: Do not add Sodium Bicarbonate.
B. Washing and Equilibration
Remove cell plate from the incubator.
Wash cells twice with the prepared warm Assay Medium.
Tip: Be gentle to avoid detaching cells.
Add final volume of Assay Medium (e.g., 180 µL for 96-well).
Incubate the cell plate at 37°C in a non-CO₂ incubator for 45–60 minutes prior to the run. This allows cell metabolism to stabilize and CO₂ to outgas.
C. Compound Preparation & Loading
Reconstitute stock compounds (Oligo, FCCP, Rot/AA) and dilute them into Assay Medium. Load the Sensor Cartridge ports as follows (volumes for XF96):
Port A (Oligomycin): Final well conc. 1.0 – 1.5 µM.
Port B (FCCP): Final well conc. 0.5 – 2.0 µM.
Note: FCCP has a bell-shaped response curve. You must titrate this for your specific cell line to find the peak (Maximal Respiration).
Port C (Rotenone/Antimycin A): Final well conc. 0.5 µM.
D. Seeding Suspension Cells (e.g., T Cells)
If using non-adherent cells (Protocol derived from STAR Protocols 3594):
Coat wells with Cell-Tak (Corning) or Poly-D-Lysine for 20 mins to adhere cells.
Plate T cells at high density (e.g., 2–5 x 10⁵ cells/well) in Assay Medium.
Centrifuge the plate at 200g for 1 min (with zero braking) to adhere cells to the bottom.
Running the Assay
Calibration: Place the Hydrated Sensor Cartridge (on the Utility Plate) into the Seahorse Analyzer. Run the "Calibrate" step (approx. 15-20 mins).
Replace Plate: Once calibration is complete, the machine will prompt you. Remove the Utility Plate and insert your Cell Culture Plate.
Standard Protocol Loop:
Mix: 3 minutes
Wait: 2 minutes
Measure: 3 minutes
Repeat loops 3 times for Basal, and after each injection.
Troubleshooting & Optimization
1. Edge Effects & Bubbles
Issue: Inconsistent data in outer wells.
Solution: Allow plates to rest at room temperature for 1 hour after seeding before moving to the incubator to promote even settling. Ensure no bubbles are trapped in the sensor cartridge ports before loading.
2. Negative OCR Values
Issue: Often caused by empty wells having higher signal than cell wells, or improper background correction.
Solution: Ensure the 4 background wells contain the exact same medium and volume as the sample wells, but no cells.
3. No Response to FCCP
Issue: Maximal respiration is not higher than basal.
Solution: The FCCP concentration might be too high (toxicity) or too low (insufficient uncoupling). Perform an FCCP titration curve (e.g., 0.5, 1.0, 2.0 µM).
4. Normalization (Crucial Step)
Raw OCR data depends on the number of cells. To compare between groups, you must normalize.
Method A (Direct Count): Use an automated imager (like Cytation) to count nuclei (Hoechst stain) immediately after the assay.
Method B (Total Protein): Lyse cells after the assay and perform a BCA protein assay.
Normalization: Convert OCR from pmol/min to pmol/min/µg protein or pmol/min/1000 cells.
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