Two-Photon Permeabilization and Calcium Measurements in Cellular Organelles

Inositol trisphosphate and cyclic ADP-ribose, main intracellular Ca²⁺ messengers, induce release from the intracellular Ca²⁺ stores via inositol trisphosphate and ryanodine receptors, respectively. Recently, studies using novel messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releasing Ca²⁺ from calcium stores in organelles other than endoplasmic reticulum (ER) have been conducted. However, technical difficulties of Ca²⁺ measurements in relatively small Ca²⁺ stores prompted us to develop a new, more sensitive, and less damaging two-photon permeabilization technique. Applied to pancreatic acinar cells, this technique allowed us to show that all three messengers – IP₃ , cADPR, and NAADP – release Ca²⁺ from two intracellular stores: the endoplasmic reticulum and an acidic store in the granular region. This chapter describes a detailed procedure of using this technique with pancreatic acinar cells.