The effect of rotenone contamination on high-resolution mitochondrial respiration experiments
Background High-resolution respirometry is commonly used in skeletal muscle research and exercise science to measure mitochondrial respiratory function in both permeabilized muscle fibers and isolated mitochondria. Due to the low throughput and high cost of the most used respirometer, the Oroboros 2...
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2025-08-01
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| author | Dale F. Taylor Jia Li Nicholas J. Saner Jia Wei Xu Yan Elizabeth G. Reisman Hanzhe Li Matthew J-C Lee Navabeh Zare Andrew Garnham Jujiao Kuang David J. Bishop |
| author_facet | Dale F. Taylor Jia Li Nicholas J. Saner Jia Wei Xu Yan Elizabeth G. Reisman Hanzhe Li Matthew J-C Lee Navabeh Zare Andrew Garnham Jujiao Kuang David J. Bishop |
| author_sort | Dale F. Taylor |
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| description | Background High-resolution respirometry is commonly used in skeletal muscle research and exercise science to measure mitochondrial respiratory function in both permeabilized muscle fibers and isolated mitochondria. Due to the low throughput and high cost of the most used respirometer, the Oroboros 2k (O2k), multiple experiments are often conducted within the same chamber in short succession. Despite this, no methodological consideration has been given for the potential contamination of inhibitors, used to investigate the contribution of specific complexes within the electron transport chain, between experiments. Methods We first assessed the potential effect of inhibitor contamination on mitochondrial respiration experiments by evaluating the ability of the currently recommended wash protocol to remove rotenone and compared its efficacy against a simplified wash protocol of sequential rinses. Secondly, we assessed the potential effect of inhibitor contamination on mitochondrial respiration measured before and after a single session of high-intensity interval exercise, with and without the use of rotenone between experiments. Results The currently recommended protocol for washing chambers was insufficient for removing rotenone. Following exercise, a decrease in mitochondrial respiration was observed exclusively in chambers exposed to rotenone between experiments. Discussion Our findings highlight an important methodological consideration regarding the measurement of mitochondrial respiratory function using high-resolution respirometry, with inhibitor contamination potentially affecting the conclusions derived from experiments conducted in close succession. Future studies investigating mitochondrial respiratory function should assess the necessity of using inhibitors such as rotenone, ensure thorough wash procedures between experiments, and explicitly report the washing protocols used. |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-08-01 |
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| spelling | doaj-art-72caf2f014a14a6281fd0d2b2fd74a0b2025-08-20T03:46:41ZengPeerJ Inc.PeerJ2167-83592025-08-0113e1987910.7717/peerj.19879The effect of rotenone contamination on high-resolution mitochondrial respiration experimentsDale F. Taylor0Jia Li1Nicholas J. Saner2Jia Wei3Xu Yan4Elizabeth G. Reisman5Hanzhe Li6Matthew J-C Lee7Navabeh Zare8Andrew Garnham9Jujiao Kuang10David J. Bishop11Institute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaMary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaInstitute for Health and Sport, Victoria University, Melbourne, Victoria, AustraliaBackground High-resolution respirometry is commonly used in skeletal muscle research and exercise science to measure mitochondrial respiratory function in both permeabilized muscle fibers and isolated mitochondria. Due to the low throughput and high cost of the most used respirometer, the Oroboros 2k (O2k), multiple experiments are often conducted within the same chamber in short succession. Despite this, no methodological consideration has been given for the potential contamination of inhibitors, used to investigate the contribution of specific complexes within the electron transport chain, between experiments. Methods We first assessed the potential effect of inhibitor contamination on mitochondrial respiration experiments by evaluating the ability of the currently recommended wash protocol to remove rotenone and compared its efficacy against a simplified wash protocol of sequential rinses. Secondly, we assessed the potential effect of inhibitor contamination on mitochondrial respiration measured before and after a single session of high-intensity interval exercise, with and without the use of rotenone between experiments. Results The currently recommended protocol for washing chambers was insufficient for removing rotenone. Following exercise, a decrease in mitochondrial respiration was observed exclusively in chambers exposed to rotenone between experiments. Discussion Our findings highlight an important methodological consideration regarding the measurement of mitochondrial respiratory function using high-resolution respirometry, with inhibitor contamination potentially affecting the conclusions derived from experiments conducted in close succession. Future studies investigating mitochondrial respiratory function should assess the necessity of using inhibitors such as rotenone, ensure thorough wash procedures between experiments, and explicitly report the washing protocols used.https://peerj.com/articles/19879.pdfOroboros O2kPermeabilized fibersProtocol developmentExerciseExercise-induced adaptationRespiration |
| spellingShingle | Dale F. Taylor Jia Li Nicholas J. Saner Jia Wei Xu Yan Elizabeth G. Reisman Hanzhe Li Matthew J-C Lee Navabeh Zare Andrew Garnham Jujiao Kuang David J. Bishop The effect of rotenone contamination on high-resolution mitochondrial respiration experiments PeerJ Oroboros O2k Permeabilized fibers Protocol development Exercise Exercise-induced adaptation Respiration |
| title | The effect of rotenone contamination on high-resolution mitochondrial respiration experiments |
| title_full | The effect of rotenone contamination on high-resolution mitochondrial respiration experiments |
| title_fullStr | The effect of rotenone contamination on high-resolution mitochondrial respiration experiments |
| title_full_unstemmed | The effect of rotenone contamination on high-resolution mitochondrial respiration experiments |
| title_short | The effect of rotenone contamination on high-resolution mitochondrial respiration experiments |
| title_sort | effect of rotenone contamination on high resolution mitochondrial respiration experiments |
| topic | Oroboros O2k Permeabilized fibers Protocol development Exercise Exercise-induced adaptation Respiration |
| url | https://peerj.com/articles/19879.pdf |
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