Fig. 2. Isoflurane and stem cell proliferation. 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. Dividing cells were labeled at the end of treatment or 6 or 24 h later by incorporation of EdU. EdU staining in a control plate (A) . EdU staining in a matched plate treated with isoflurane, 2.8%, for 6 h. Hoechst (33342) nuclear staining (blue); EdU staining (Alexa Fluor 488) (green ); nestin staining (Alexa Fluor 555) (red ) (B) . Isoflurane, 0.7%, had no effect at any time point (P = 0.60) (C) . In contrast, isoflurane, 1.4 (P = 0.004) (D) and 2.8% (P = <0.001) (E), decreased the number of EdU-positive cells; the change lasted at least 24 h with isoflurane, 2.8%. Data at each concentration were analyzed by two-way ANOVA, using treatment condition and time posttreatment as the variables. Data are expressed as mean ± SD for N = 8 wells per treatment condition (eight images per well using a 20× objective) at each time point. *P < 0.001, 24 h after isoflurane versus end of treatment and 6 h after treatment; #P < 0.01, $$P < 0.001 end of isoflurane and 6 h after treatment versus control; $P < 0.01 24 h after isoflurane treatment versus control.