Aug 20, 2024

Public workspaceIsolated Brain Mitochondria Respiration protocol

DOI
dx.doi.org/10.17504/protocols.io.bp2l62z4zgqe/v1
  • 1California Institute of Technology;
  • 2UCLA Metabolomics Center, David Geffen School of Medicine at the University of California, Los Angeles, CA, USA
Livia Hecke Morais
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DOI: dx.doi.org/10.17504/protocols.io.bp2l62z4zgqe/v1
Protocol Citation: Livia Hecke Morais, Linsey Stiles 2024. Isolated Brain Mitochondria Respiration protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62z4zgqe/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: August 20, 2024
Last Modified: August 20, 2024
Protocol Integer ID: 106024
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Abstract
Brain Mitochondria Respiration protocol. Assay developed by Dr. Linsey Stiles at the UCLA Metabolomics Center, David Geffen School of Medicine at the University of California, Los Angeles, CA, USA
Preparation
Preparation
The day prior to the Seahorse assay, a Seahorse cartridge needs to be hydrated overnight.

All isolated brain mitochondria samples are prepared in Mitochondrial Respiration Buffer (MAS) prepared with containing: 220 mM mannitol, 70 mM sucrose, 5 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, and 0.1% (w/v) fatty acid-free BSA.
Load the Seahorse Cartridge
Load the Seahorse Cartridge
Prepare the compounds in MAS to be loaded into the Seahorse cartridge ports. Final concentrations in the well were:
a. 4 mM ADP
b. 3 µM oligomycin
c. 4 µM FCCP
d. 2 µM rotenone and antimycin A
Load 20 µL per port in the Seahorse cartridge with a multichannel pipet
Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
The prepared Seahorse cartridge needs to be calibrated prior to loading the sample plate.
1. Prepare template with plate map and running program. An example of an isolated mitochondria running protocol:
Command Time (minutes) Port Repeat
Calibrate 18
Mix 2 2
Time Delay 2
Mix 0.5 1
Measure 2
Mix 1
Inject                                                                           A (ADP + substrates)
Mix 0.5 2
Measure 2
Mix 1
Inject                                                                                B (Oligomycin)
Mix 0.5 2
Measure 2
Mix 1
Inject                                                                                 C (FCCP)
Mix 0.5 2
Measure 2
Mix 1
Inject                                                   D (Antimycin A)
Mix 0.5 2
Measure 2
End Program
2. Start the Seahorse Run
3. Load the cartridge for calibration
1. Prepare mitochondria sample dilutions based on the protein concentration of each sample
a. Samples preparation includes 10X substrate, isolated mitochondria, and MAS Buffer for the desired number of wells
2. Load 20µL of the mitochondria sample per well of the XF96 microplate
3. Centrifuge the plate at 2,100 x g for 5 minutes
4. Stop the centrifugation without a break
5. Add 130 of MAS per well after centrifugation
6. Load the Seahorse plate into the instrument