Dataset Information

Quantitative microplate-based respirometry with correction for oxygen diffusion.

ABSTRACT: Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O(2) is consumed by the specimen, atmospheric O(2) leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O(2) but also store substantial amounts of gas. O(2) flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O(2)] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O(2)]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems.

SUBMITTER: Gerencser AA

PROVIDER: S-EPMC2727168 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Quantitative microplate-based respirometry with correction for oxygen diffusion.

Gerencser Akos A AA Neilson Andy A Choi Sung W SW Edman Ursula U Yadava Nagendra N Oh Richard J RJ Ferrick David A DA Nicholls David G DG Brand Martin D MD

Analytical chemistry 20090801 16

Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O(2) is consumed by the specimen, atmospheric O(2) leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeab ...[more]

PMID: 19555051

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Project description:Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.

Dataset Information

Quantitative microplate-based respirometry with correction for oxygen diffusion.

Publications

Quantitative microplate-based respirometry with correction for oxygen diffusion.

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