Measuring Ca²⁺ Release Evoked by Cyclic ADP-Ribose

Abstract

As a ubiquitous second messenger, the Ca²⁺ mobilizing activity of cyclic ADP-ribose (cADPR) has been observed in many different cell types. The measurement of Ca²⁺ release evoked by cADPR comprises several practical challenges. At physiological pH, cADPR has a net negative charge and it therefore cannot cross the cell membrane in cells that lack a suitable cADPR-transporting system. Thus, either the plasma membrane must be permeabilized or microinjection must be used to deliver cADPR to the cytosol. In this article, two methods for cADPR delivery (using permeabilized cells or microinjection) are explained step-by-step. Because most of our work has been performed using the Jurkat T-lymphoma cell line, the methods are tailored for this specific cell type. For other cell types, the procedures may need to be adapted.