Measuring Mitochondrial Respiration in Previously Frozen Biological Samples, 2020, Shirihai et al

[Andy]

Measuring oxygen consumption allows for the role of mitochondrial function in biological phenomena and mitochondrial diseases to be determined. Although respirometry has become a common approach in disease research, current methods are limited by the necessity to process and measure tissue samples within 1 hr of acquisition. Detailed by Acin‐Perez and colleagues, a new respirometry approach designed for previously frozen tissue samples eliminates these hurdles for mitochondrial study. This technique allows for the measurement of maximal respiratory capacity in samples frozen for long‐term storage before testing. This protocol article describes the optimal tissue isolation methods and the combination of substrates to define electron transport chain function at high resolution in previously frozen tissue samples. © 2020 The Authors.

Basic Protocol 1: Sample collection, storage, and homogenization for previously frozen tissue respirometry

Basic Protocol 2: Running a Seahorse respirometry assay using previously frozen tissue samples

Basic Protocol 3: Normalization to mitochondrial content for previously frozen tissue respirometry

Open access, https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpcb.116

Freeze‐thawing impairs mitochondrial respiration by disrupting and permeabilizing the mitochondrial inner membrane, which releases electron carriers that support the electron transport chain (Figure 2C). However, the interaction of electron transport complexes (supercomplexes) remains intact under freeze‐thawing conditions, allowing for integrated electron transport as seen in fresh tissues. This article, therefore, provides instructions to rescue the defects induced by freeze‐thawing, as well as to control the entry of electrons into the electron transport system by providing different electron carriers. As our procedure allows for the specific measurement of oxygen consumed by complex IV, mitochondrial isolation is no longer necessary, allowing for the use of homogenates to measure mitochondrial function, significantly simplifying tissue preparation (described in Basic Protocol 1).