Jan 10, 2026
Assessment of Mitochondrial OCR For SH-SY5Y Cell using Agilent Seahorse XFe Cell Mito Stress Test
- 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
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Assessment of Mitochondrial OCR For SH-SY5Y Cell using Agilent Seahorse XFe Cell Mito Stress Test . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qdypl1y/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: October 13, 2024
Last Modified: January 10, 2026
Protocol Integer ID: 109767
Keywords: Mitochondrial function, Mitochondrial OCR, Seahorse assay, using agilent seahorse xfe cell mito stress test, agilent seahorse xfe cell mito stress test, assessment of mitochondrial function, assessment of mitochondrial ocr, indicator of mitochondrial function, mitochondrial ocr, mitochondrial function, sy5y cell, oxygen species, effects of iron supplementation, oxygen consumption rate, iron supplementation
Abstract
This protocol describes the assessment of mitochondrial function in SH-SY5Y cells using the Agilent Seahorse XFe Cell Mito Stress test, which measures the oxygen consumption rate (OCR) as an indicator of mitochondrial function. Additionally, it evaluates the effects of iron supplementation and reactive oxygen species (ROS) inhibition.
Troubleshooting
Cell Seeding and Treatment
2d
Seed 80,000 cells (SH-SY5Y) in 180 μl media in each well of a Seahorse XFe96 microplate.
Note: Leave well A blank empty as background control.
For iron supplementation, treat cells with 0.2 mg/mL ferric ammonium citrate (FAC)
For ROS inhibition, treat cells with 250 nM Liproxstatin-1
Incubate the plate at 37 °C with 5% CO2 for 48:00:00
2d
Preparation of Sensor Cartridge
Hydrate the sensor cartridge
The day prior to measuring OCR, hydrate the sensor cartridge with Seahorse XF Calibrant 200 µL /well and 400 µL PBS/ edge well and keep the sensor cartridge at 37 °C in a non-CO2 incubator overnight
Preparing the Assay Medium
1h
On the day of the assay, replace the growth medium with pre-warmed assay medium (180 µL ) containing Seahorse XF base medium, 1 mM pyruvate, and 20 mM glucose.
Incubate the microplate in a non-CO2 incubator at 37 °C for 01:00:00 to equilibrate the temperature and pH.
1h
Preparing Modulating Agents
In the meantime, prepare following modulating agents (10X) in assay medium for injection:
| A | B | C | D | |
| Port Loading Design | 10X concentration | Add to Port volume | Final well concentration (µM) | |
| Port A: Oligomycin | 10 μM | 20 μL | 1 µM | |
| Port B：2,4-Dinitrophenol (DNP) | 500 µM | 25 μL | 50 µM | |
| Port C：Rotenone | 5 µM | 27 μL | 0.5 µM |
Load 10X concentration of agents into port of the cartridge (From step 2.1 )
Calibrating the Analyzer
15m
Place the loaded sensor cartridge into analyzer and run the program. The calibration usually takes 00:15:00 .
15m
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.