Measurement for CCCP-induced thermogenesis in rat neonatal cardiomyocytes. (A) Left: Bright-field image of rat neonatal cardiomyocytes and contaminating fibroblasts (indicated by “F”). Right: Fluorescence image of EuTTA for the cells on the left on a RT nanosheet. (B) Time course of changes in the EuTTA/Rh101 ratio of RT nanosheets with cardiomyocytes upon application of 10 µM CCCP (red) or 0.1% DMSO (black). Values were corrected by those obtained in the medium (see Materials and methods for details). Number of cells, 18 (n = 3) for both CCCP and DMSO. The horizontal black line indicates the period during which CCCP or DMSO was present. Calibration bar was calculated from the temperature sensitivity of RT nanosheets (−3.35%/°C, cf. Fig. 1 D). Data shown as mean ± SEM (C) Left: Bright-field image of rat neonatal cardiomyocytes and contaminating fibroblasts (indicated by “F”) on a glass-bottom dish. Middle and right: Fluorescence images of ER thermo yellow and ER tracker green for the cells on left, respectively. (D) Time course of change in the F.I. of ER thermo yellow (purple) and ER tracker green (green) upon an increase in temperature by IR laser irradiation for 2 s (as indicated by “Heating”). F.I. around the heated center (9,331 µm2) was measured. Data shown as mean ± SEM (n = 4). (E) Time course of changes in the F.I. of ER thermo yellow in cardiomyocytes before and after application of 10 µM CCCP (red) or 0.1% DMSO (black). (F) Same as in E for ER tracker green. In E and F, number of cells, 13 (n = 2) and 8 (n = 2) for CCCP and DMSO, respectively. Data shown as mean ± SEM. (G) Time courses of changes in the F.I. of ER thermo yellow in cardiomyocytes preincubated with 10 µM rotenone (blue) or 0.1% DMSO (black) for 30 min and then stimulated by 10 µM CCCP. (H) Same as in G for ER tracker green. In G and H, number of cells, 11 (n = 2) for both rotenone and DMSO. Data shown as mean ± SEM.
