Jan 11, 2026

Public workspaceMeasurement of Oxygen Consumption Rate (OCR) in iPSC-Derived Neurons and iPSCs Using Seahorse XFe96 Analyzer

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l214o3g1y/v1
  • Ali Ghoochani1,2,3,4,
  • Monther Abu-Remaileh1,2,3,4
  • 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
  • 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
  • 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
  • 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
  • asap
Ali Ghoochani
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.4r3l214o3g1y/v1
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Measurement of Oxygen Consumption Rate (OCR) in iPSC-Derived Neurons and iPSCs Using Seahorse XFe96 Analyzer. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l214o3g1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 23, 2025
Last Modified: January 11, 2026
Protocol Integer ID: 223019
Keywords: mitochondrial oxygen consumption rate, measurement of oxygen consumption rate, using seahorse xfe96 analyzer, oxygen consumption rate, seahorse xfe96 analyzer this protocol, seahorse xfe96 analyzer, ipsc, ocr, undifferentiated ipsc, derived neuron
Abstract
This protocol describes the procedure to measure mitochondrial oxygen consumption rate (OCR) in iPSC-derived neurons and undifferentiated iPSCs using the Seahorse XFe96 Analyzer.
Materials
-Seahorse XFe96 microplates (Agilent Technologies)
- Poly-D-Lysine (for neurons)
- rhLaminin (for iPSCs)
- Seahorse XF Base Medium (Agilent Technologies)
- DMEM/F12 (Thermo Fisher Scientific)
- Neurobasal-A (Thermo Fisher Scientific)
- MEM Non-Essential Amino Acids (Thermo Fisher Scientific)
- GlutaMAX (Thermo Fisher Scientific)
- N2 Supplement (Thermo Fisher Scientific)
- B27 Supplement with/without antioxidant (Thermo Fisher Scientific)
- NT-3 (10 ng/ml)
- BDNF (10 ng/ml)
- Mouse Laminin (1 µg/ml)
- Doxycycline hydrochloride (2 µg/ml)
- Pyruvate (1 mM)
- Glucose (20 mM)
- Oligomycin (1 µM final)
- 2,4-Dinitrophenol (DNP, 50 µM final)
- Rotenone (0.5 µM final)
- StemFlex‱ Medium (Thermo Fisher Scientific)
- Seahorse XFe Report Generator
- Protein quantification reagents
Troubleshooting
Maintain iPSCs
Culture iPSCs in StemFlex™ Medium on rhLaminin-coated plates (2.5 μg/mL in 1x DPBS).
Replace medium every other day.
Initiate Pre-Differentiation (Day -3 to 0)
Detach iPSCs using ReLeSR™ and centrifuge to pellet.
Resuspend in N2 Pre-Differentiation Medium:

  • Knockout DMEM/F12
  • 1x MEM Non-Essential Amino Acids
  • 1x N2 Supplement
  • 10 ng/mL NT-3
  • 10 ng/mL BDNF
  • 1 μg/mL Mouse Laminin
  • 10 μM Thiazovivin
  • 2 μg/mL doxycycline
Seed 1.5 × 10⁶ cells/well in rhLaminin-coated 6-well plates with 2 mL/well of medium.
Pre-Differentiation (Day -3 to 0)
Maintain cells without medium change.
On Day 0, dissociate with Accutase and centrifuge.
Neuronal Differentiation and Maturation (Days 0–21)
Plate 5 × 10⁴ pre-differentiated cells per well into Poly-D-Lysine-coated Seahorse XFe96 microplates.
Critical
Culture in Classic Neuronal Medium (1:1 DMEM/F12 and Neurobasal-A) supplemented with:

  • 1x MEM NEAA
  • 0.5x GlutaMAX
  • 0.5x N2
  • 0.5x B27 with/without antioxidant (Based on the experiment)
  • 10 ng/mL NT-3
  • 10 ng/mL BDNF
  • 1 μg/mL Mouse Laminin
  • 2 μg/mL doxycycline

Day 7: Half-medium change, no doxycycline.
Day 14: Repeat half-medium change with same composition (no doxycycline).
Day 21: Cells ready for Seahorse assay.
Preparation for Seahorse Assay
Preparation of Sensor Cartridge (a day before experiment):

Hydrate the sensor cartridge:

The day prior to measuring OCR, hydrate the sensor cartridge with Seahorse XF Calibrant Amount200 µL /well and Amount400 µL PBS/ edge well and keep the sensor cartridge at Temperature37 °C in a non-CO2 incubator overnight​
Critical
Preparing the Assay Medium
On the day of the assay, replace the growth medium with pre-warmed assay medium (Amount180 µL ) containing Seahorse XF base medium, 1 mM pyruvate, and 20 mM glucose.

Incubate the microplate in a non-CO2 incubator at Temperature37 °C for Duration01:00:00 to equilibrate the temperature and pH.
Preparing Modulating Agents
In the meantime, prepare following modulating agents (10X) in assay medium for injection:

ABCD
Port Loading Design10X concentrationAdd to Port volume Final well concentration (µM)
Port A: Oligomycin 10 μM20 μL1 µM
Port B：2,4-Dinitrophenol (DNP)500 µM 25 μL50 µM
Port C：Rotenone5 µM27 μL0.5 µM

Load 10X concentration of agents into port of the cartridge (From step 13 )
Calibrating the Analyzer
Place the loaded sensor cartridge into analyzer and run the program. The calibration usually takes Duration00:15:00 .
Measuring the OCR
After calibration is done, transfer the Seahorse XFe96 cell microplate (from step 3.1) to the analyzer and press "continue"
Post-Assay Procedures
Protein Normalization:
After the assay, aspirate the assay medium and lyse the cells with a suitable lysis buffer and measure protein concentration using a BCA assay.
Use this data for normalizing the OCR results.
OCR Measurement in iPSCs (Undifferentiated)
Plate 5 × 10⁴ iPSCs per well into rhLaminin-coated Seahorse XFe96 microplates in StemFlex™ Medium.
Incubate 48 h at 37°C, 5% CO₂.
Seahorse Assay :



follow same steps as in Steps 13–19 above.