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

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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.
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