Measurements of [Ca ²⁺ ] _cyt and temperature of the sarcoplasmic reticulum in FDB fibers during isoflurane application. (A) Bright-field (lower left panels) and fluorescence images of FDB fibers isolated from WT (left) and R2509C-Het (right) mice following co-staining with Cal-520 and ERthermAC. Lower right panels show higher magnification of the area depicted by white-outlined squares in upper right panels. (B) Time course of changes in the relative fluorescence intensity of Cal-520 (top) and ERthermAC (bottom) in WT (left) and R2509C-Het (right) cells. Gray lines represent individual cells. Thick colored lines represent average responses. Isoflurane (1%) was applied at the time points indicated by yellow horizontal bars. Photobleaching of ERthermAC was corrected by a single-exponential fitting (Arai et al., 2014). See Fig. S5 for raw data. (C) Maximum values (F_max/F₀) in the relative fluorescence intensity of Cal-520 (left) and minimum values (F_min/F₀) in the relative fluorescence intensity of ERthermAC (right) during isoflurane application. WT: n = 17, N = 4; R2509C-Het: n = 10, N = 4. Values are means ± SD. Statistical significance was determined using a one-way ANOVA with Tukey’s test. “n” indicates the number of FDB fibers, and “N” the number of animals.