Quantitative ratiometric Ca2+ imaging to assess cell viability

Viability of cells is strongly related to their Ca2+ homeostasis. Ca2+ signal fluctuations can be on a slow time scale, e.g., in non-excitable cells, but also in the range of tens of milliseconds for excitable cells, such as nerve and muscle. Muscle fibers respond to electrical stimulation with Ca2+ transients that exceed their resting basal level about 100 times. Fluorescent Ca2+ dyes have become an indispensable means to monitor Ca2+ fluctuations in living cells online. Fluorescence intensity of such “environmental dyes” relies on a buffer-ligand interaction which is not only governed by laws of mass action but also by binding and unbinding kinetics that have to be considered for proper Ca2+ kinetics and amplitude validation. The concept of Ca2+ dyes including the different approaches using ratiometric and non-ratiometric dyes, the way to correctly choose dyes according to their low-/high-affinity properties and kinetics as well as staining techniques, and in situ calibration are reviewed and explained. We provide detailed protocols to apply ratiometric Fura-2 imaging of resting Ca2+ and Ca2+ fluctuations during field-stimulation in single isolated skeletal muscle cells and how to translate fluorescence intensities into absolute Ca2+ concentrations using appropriate calibration techniques.