Apr 07, 2026
Seahorse assay for measuring oxygen consumption rate in iPSC-derived glutamatergic neurons
- Tzu-Chieh Huang1,2,
- Marisa DeCicco3,4,
- Hongying Shen3,4,
- Kristen Brennand1,2
- 1Department of Psychiatry, Division of Molecular Psychiatry, Yale University School of Medicine, New Haven, CT 06511;
- 2BD2: Breakthrough Discoveries for thriving with Bipolar Disorder, Santa Monica, CA;
- 3Department of Cellular 26 Molecular Physiology, Yale School of Medicine, New Haven, CT, 06511;
- 4Systems Biology Institute, Yale West Campus, West Haven, CT 06516
Protocol Citation: Tzu-Chieh Huang, Marisa DeCicco, Hongying Shen, Kristen Brennand 2026. Seahorse assay for measuring oxygen consumption rate in iPSC-derived glutamatergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jw1dvqe/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: April 07, 2026
Last Modified: April 07, 2026
Protocol Integer ID: 314600
Keywords: measuring oxygen consumption rate, measurement of oxygen consumption rate, oxygen consumption rate, seahorse xfe24 analyzer, quantification of basal respiration, glutamatergic neuron, glutamatergic neurons this protocol, derived glutamatergic neuron, respiratory capacity, basal respiration, maximal respiration, total protein content, total protein content from cell lysate, seahorse, ocr value
Funders Acknowledgements:
BD2 Discovery
Abstract
This protocol describes the measurement of oxygen consumption rate (OCR) in human iPSC-derived glutamatergic neurons (iGLUTs) using the Seahorse XFe24 Analyzer (Agilent). OCR values are normalized to total protein content from cell lysates to account for differences in cell number. The expected outcomes include quantification of basal respiration, ATP production, maximal respiration, and spare respiratory capacity.
Materials
- XF24 microplates (Agilent, 100777-004)
- Seahorse XF DMEM (Agilent, 103575-100)
- Seahorse XF 1.0 M glucose solution (Agilent, 103577-100)
- Seahorse XF 100 mM pyruvate solution (Agilent, 103578-100)
- Seahorse XFe24 Analyzer (Agilent)
- Oligomycin A (Sigma-Aldrich, 75351)
- Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (Sigma-Aldrich, C2920)
- Rotenone (Sigma-Aldrich, R8875)
- Antimycin A (Sigma-Aldrich, A8674)
- M-PER™ Mammalian Protein Extraction Reagent (ThermoFisher, 78501)
- cOmplete™ Mini Protease Inhibitor Cocktail (Sigma-Aldrich, 11836153001)
- PhosSTOP™ (Sigma-Aldrich, 4906845001)
- Pierce™ Dilution-Free™ Rapid Gold BCA Protein Assay (ThermoFisher, A55860)
Procedure
Seed 1.65x10^5^ Day 5 iPSC-derived glutamatergic neurons (iGLUTs) into each well of a XF24 microplate and continue differentiation until Day 21 for the mitochondrial stress test.
Prior to the assay, remove most of the culture medium, leaving only 50 µL per well, and add 1000 µL pre-warmed Seahorse XF DMEM supplemented with 25mM glucose and 0.23mM pyruvate.
Incubate the Seahorse plate at 37 °C in a non-CO2 incubator for 1 hour.
After the 1-hour incubation, replace the assay medium with 500 µL of fresh assay medium for the Seahorse assay.
Measure the plate using Seahorse XFe24 Analyzer with the standard Mito Stress Test program. The program includes three sequential injections of mitochondrial inhibitors: 1.5 µM oligomycin, 1.5 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, and a mixture of 0.5 µM rotenone and 0.5 µM antimycin A.
After recording the oxygen consumption rate, lyse the cells with 30 µL of M-PER™ Mammalian Protein Extraction Reagent containing cOmplete™ Mini Protease Inhibitor Cocktail and PhosSTOP™, according to the manufacturer’s instructions.
Measure total protein concentration using Pierce™ Dilution-Free™ Rapid Gold BCA Protein Assay.
Normalize oxygen consumption rates to total protein content.