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Updated: Feb 19, 2026
08:33
High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells
Published on: February 8, 2017
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The effect of respiration buffer composition on mitochondrial metabolism and function.
Lucas C Wollenman1,2, Matthew R Vander Ploeg1, Mackinzie L Miller3,4
1Department of Physiology, Michigan State University, East Lansing, MI, United States of America.
Plos One
|November 2, 2017
Summary
Choosing the right respiration buffer is crucial for accurate mitochondrial studies. Buffers without high chloride concentrations, like K-lactobionate or K-gluconate, significantly enhance ADP-stimulated respiration rates compared to standard KCl buffers.
Area of Science:
- Mitochondrial physiology and bioenergetics.
- Biochemical analysis of cellular respiration.
Background:
- Accurate functional studies of isolated mitochondria depend on appropriate respiration buffer selection.
- Variations in buffer composition can significantly alter respiration rates, hindering inter-study comparisons.
- The ideal buffer should maximize ADP-stimulated respiratory rates and minimize substrate transport limitations.
Purpose of the Study:
- To evaluate various respiration buffers and substrate combinations for optimal mitochondrial function.
- To determine buffer conditions that maximize ADP-stimulated and uncoupled respiration.
- To identify the impact of buffer composition, specifically chloride concentration, on mitochondrial respiratory states.
Main Methods:
- Isolated cardiac guinea pig mitochondria were used.
- Oxygen consumption was measured using an Oroboros Oxygraph-2k.
- Respiration was assessed in leak, ADP-stimulated, and FCCP-uncoupled states across different buffer compositions (KCl-based vs. K-lactobionate/K-gluconate-based) and substrate combinations.
Main Results:
- Buffers B2, B3, and B4 (lacking high chloride) showed increased respiration rates compared to the standard KCl buffer (B1).
- Specifically, B2, B3, and B4 demonstrated average increases of 16% (leak), 26% (ADP-stimulated), and 35% (uncoupled) respectively, relative to B1.
- Chloride appears to partially inhibit key mitochondrial transporters like the adenine nucleotide translocase and dicarboxylate carrier.
Conclusions:
- Buffers utilizing K-lactobionate or K-gluconate are superior to standard KCl buffers for maximizing ADP-stimulated mitochondrial respiration.
- The absence of high chloride concentrations in buffers is key to enhancing mitochondrial respiratory function.
- These findings provide critical guidance for selecting optimal buffers in mitochondrial research to improve data reproducibility and comparability.
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