Fig. 4. Effect of isoflurane on cell death. With the use of matched plates, neural stem cells were exposed to control conditions or isoflurane, 0.7, 1.4, or 2.8%, for 6 h. The percentage of dead cells was determined by propidium iodide (PI) staining at the end of treatment or 6 or 24 h later. Image of PI-stained cells in a control plate (A) . Image of PI staining in a matched plate 24 h after isoflurane, 2.8%(B) . Hoechst (33342) nuclear staining (blue ); nestin staining (Alexa Fluor 488) (green ); PI (pink ). PI-positive cells (arrows ). Compared with the corresponding control, isoflurane, 0.7 (C) , 1.4 (D) , and 2.8%(E) , had no effect on the percentage of cells that stained positive for PI, but there was an effect of time after treatment, with a larger percentage of cells staining positive for PI 6 and 24 h after treatment with isoflurane, 1.4 and 2.8%, compared with the end of treatment time point. Because the data failed normality testing, results were analyzed using Kruskal-Wallis one-way ANOVA followed by Dunn's testing for multiple comparisons. Data are expressed as median (interquartile range) for N = 12 wells per treatment condition (eight images per well using a 20× objective) at each time point.