Signal Transduction Protocols

Volume 41 of the series Methods in Molecular Biology™ pp 203-213

Measurement of Intracellular Free Calcium Ion Concentration in Cell Populations Using Fura-2

  • Philip A IredaleAffiliated withDepartment of Physiology and Pharmacology, Queen’s Medical Centre
  • , John M. DickensonAffiliated withDepartment of Physiology and Pharmacology, Queen’s Medical Centre

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The early methods for calcium measurement involved microinjection of calcium-sensitive proteins, such as aequorin or obelin, into large cells (1, 2) or the use of microelectrodes (3). Both techniques are still employed, however, with much improved sensitivity allowing investigation of a greater range of cell types. In the early 1980s, the “Null Point method” was introduced (4), which involved addition of a metallochromic calcium indicator (Arsenazo III) to cells permeabilized with digitonin. Using this technique, the accumulation and release of calcium from intracellular stores could be recorded. A major advance in calcium measurement was made when Tsien and his colleagues (5, 6) introduced fluorescent calcium indicators. The first to be used was quin-2: its structure was based on the novel calcium chelator 1,2-bis-(O-aminophenoxyl-ethane-N, N,N′,N′-tetraacetic acid (BAPTA) (7, 8), a double aromatic analog of EGTA. The major problem of inducing a hydrophilic polycarboxylate anion to cross the plasma membrane was overcome by the addition of an acetoxymethyl ester group (AM), thus producing a lipophilic, membrane-permeant molecule (quin-2 AM) that, once within the cytoplasm, was subject to attack by intracellular enzymes, which cleaved the ester bond and left the calcium-sensitive free acid trapped within the cell (5). A number of improved calcium indicators have since been developed (e.g., fura-2, indo-1, and fluo-3), but the basic principles of dye loading and continuous calcium reporting remain the same.