Coverslips in 12-well plates were placed in identical air-tight, humidified chambers (Billups-Rothenberg, Del Mar, CA, USA) as previously described [43 (link)]. Isoflurane (Baxter Healthcare Cooperation, Deerfield, IL, USA) or sevoflurane (AbbVie Inc., North Chicago, IL, USA) were delivered using an agent-specific, calibrated inline vaporizer (SuperaVet, Vaporizer Sales and Services Inc., Rockmart, GA, USA), and were diluted in 5% CO2/95% O2 carrier gas. Controls for these experiments received 5% CO2/95% O2 carrier gas only. There was a 15-min equilibration period, which was required to achieve the correct concentration of Isoflurane or sevoflurane as measured by a 5250 RGM gas analyzer (Datex-Ohmeda, Madison, WI, USA). Then the sealed chambers were placed in an incubator to maintain temperature at 37 °C for the duration of anesthesia exposure. Isoflurane/sevoflurane concentration was periodically measured at the end of the experimental period to verify that it was appropriately maintained throughout the exposure.
The propofol exposure was done by adding pure 2,6-diisopropylphenol (Sigma Aldrich, Saint Louis, MO, USA) (1 nM, 2 nM, 4 nM) into experiment wells, and incubated at 37 °C for the duration of anesthesia exposure. The exposure was terminated by removing all the media and by adding a combination of previously-removed media without propofol and fresh media.
The propofol exposure was done by adding pure 2,6-diisopropylphenol (Sigma Aldrich, Saint Louis, MO, USA) (1 nM, 2 nM, 4 nM) into experiment wells, and incubated at 37 °C for the duration of anesthesia exposure. The exposure was terminated by removing all the media and by adding a combination of previously-removed media without propofol and fresh media.
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