Quantitative Ratiometric Ca²⁺ Imaging to Assess Cell Viability

Abstract

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.