Calcium Signaling Protocols

Anish Patel, Robert A. Hirst, Charlotte Harrison, Kazuyoshi Hirota, David G. Lambert

Use of Fura-2 in whole cell suspensions to measure changes in intracellular Ca(2+) is probably one of the simplest, yet most widely used protocols described in this volume. Whole cell suspensions are loaded with Fura-2 and then placed into a cuvette-based fluorimetric system (measuring 510 nm emission at alternating 340/340 nm excitation). Cells can be stimulated with agonists and antagonists to enable temporal response profiling and concentration-response curves to be constructed. The protocol can be used for a wide range of cells including those transfected with Ca(2+)-signaling proteins, e.g., receptors and channels. Loading characteristics and the need for agents to retain loaded dye (e.g., probenecid) need to be determined empirically. Calibration of whole cell suspensions to convert the fluorescent signal into Ca(2+) is simply performed using Triton-X lysis (to determine R (max)) and EGTA chelation (to determine R (min)).