Title |
Lung tissue bioenergetics and caspase activity in rodents
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Published in |
BMC Research Notes, January 2013
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DOI | 10.1186/1756-0500-6-12 |
Pubmed ID | |
Authors |
Ahmed R Alsuwaidi, Mohammed T Alsamri, Ali S Alfazari, Saeeda Almarzooqi, Alia Albawardi, Aws R Othman, Thachillath Pramathan, Stacey M Hartwig, Steven M Varga, Abdul-Kader Souid |
Abstract |
This study aimed to establish a suitable in vitro system for investigating effects of respiratory pathogens and toxins on lung tissue bioenergetics (cellular respiration and ATP content) and caspase activity. Wistar rats and C57Bl/6 mice were anesthetized by sevoflurane inhalation. Lung fragments were then collected and incubated at 37°C in a continuously gassed (with 95% O2:5% CO2) Minimal Essential Medium (MEM) or Krebs-Henseleit buffer. Phosphorescence O2 analyzer that measured dissolved O2 concentration as a function of time was used to monitor the rate of cellular mitochondrial O2 consumption. Cellular ATP content was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence. Lung histology and immunostaining with anti-cleaved caspase-3 antibody were also performed. |
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