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Informed consent was obtained from each patient before commencing experiments.
Mice were single-housed for 4 weeks before commencing experiments to ensure surgical recovery and optimized Cre-mediated recombination.
Prior to commencing experiments on fcDNA, we evaluated the performance of the methylation enrichment protocol with low (100 ng) DNA sample amounts, and developed a modified high-stringency protocol to limit the amount of non-specific DNA binding.
Reproducible responses were obtained to 40 mM KCl and 30 nM 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U46619, Tocris) and endothelial function was assessed by the addition of 10 μM acetylcholine before commencing experiments.
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The initial tension was adjusted to 1.5 g and an equilibration period of 90 min was allowed before commencing the experiments.
Before commencing the experiments, the participants were instructed to stop looking at the rubber hand and start looking at the knife as soon as it was presented to make sure that they perceived the threat.
Ten minutes before commencing the experiments, each bee was checked for intact PER by touching one antenna with a toothpick that had been dipped into 1M sucrose solution without allowing feeding.
The animals were allowed to acclimatize for 5 days before commencing the experiments.
After 48 h, cells were loaded with Fura-Red (3 µM) for 50 min before commencing imaging experiments.
Animals (n = 5 sham and n = 5 sepsis) were fed with a specific manganese-reduced diet over a four-week period prior to commencing the experiments (to avoid interference by the near-infrared properties of manganese).
All animals were introduced to hydrated diet (prepared as described above) 24 36 h before commencing the experiment.
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