Methods to Measure Intracellular Ca2+ Concentration Using Ca2+-Sensitive Dyes

Abstract

Ca²⁺ ion is universally considered the most versatile second messenger responsible for decoding and regulating the majority of the signaling pathways within the cell. The study of intracellular Ca²⁺ concentration ([Ca²⁺]_i) dynamics is consequently of primary importance for the interpretation of cellular biology. This chapter will present a relatively simple, largely diffused, and nevertheless robust method to measure variations of [Ca²⁺]_i by the use of the Ca²⁺-sensitive chemical dye Fura-2. A general protocol for the assessment of [Ca²⁺]_i in adherent cells, applicable to a variety of cell systems, will be first presented. Then, the implementation of Fura-2 to detect [Ca²⁺]_i in two specific cell types, namely, human adrenocortical cells and primary skin fibroblasts, will be discussed in more particulars. Finally, the procedure to monitor Ca²⁺ influx through the plasma membrane using Fura-2 will be described.

Keywords: Adherent cells; Fura-2; Intracellular calcium measurements; Ratiometric calcium indicator.