Fig. 1.

Staining adult ventricular myocytes with SRB. (A) Fluorescence image of rat and mouse ventricular myocytes stained with SRB and Hoechst 33342, and excited sequentially on a confocal system with open pinhole. (B) Relationship between total SRB fluorescence collected within the cell outline (a measure of volume) and cell area in the x-y plane (i.e. a measure that ignores cell thickness in the z-direction). Each color/shade denotes data from a separate batch of cells: grayscale represent rat myocytes (N = 221, 10 batches); colors represent mouse myocytes (N = 73, 5 batches). (C) Total SRB fluorescence in the non-nuclear cytoplasmic area (red) rises near-linearly with cell area, whereas mean SRB signal in nuclear areas is independent of cell size, making the latter a suitable reference for ratiometric analyses (mean ± SEM; N-5-52 cells per bin) (D) Ratio of extra-nuclear to nuclear SRB signal, plotted as a function of cell area. This ratiometric analysis improves correlation (Pearson coefficient R²) and corrects for batch-to-batch differences in extraneous variables such as imaging settings. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)