Fura 2-AM

Fura 2-AM is a well-established compound used in biological research for measuring the concentration of free calcium ions ([Ca2+]) within living cells. It operates as a divalent metal ion chelator, altering its fluorescence properties upon complexation with calcium ions. This allows researchers to indirectly infer [Ca2+] using the law of mass action. While primarily recognized for its role in indicating [Ca2+], Fura 2-AM also competes with endogenous calcium buffers, which can be exploited to estimate the properties of these buffers and to measure calcium fluxes across cellular membranes1.

The mechanism of action of Fura 2-AM involves its initial permeation into the cell in an esterified form. Cellular esterases then hydrolyze Fura 2-AM to release Fura-2, the active form of the dye that binds to free calcium ions. The binding of Fura-2 to [Ca2+] results in a change in its fluorescence, which can be measured using a fluorescence ratio method. This change is used to estimate the concentration of [Ca2+] within the cell. However, the hydrolysis process is not always complete, and other metabolites, as well as unhydrolyzed Fura 2-AM, may be present within the cell, which can affect the accuracy of the measurements3.

Human Skeletal Muscle Research

In the field of human skeletal muscle research, Fura 2-AM has been used to estimate myoplasmic [Ca2+] in both normal and diseased muscle fibers. The dye has proven successful in measuring resting levels and changes in [Ca2+] in response to drugs like halothane and caffeine, which is particularly relevant in the study of conditions such as malignant hyperthermia2.

Neurobiology

In neurobiology, Fura 2-AM has been utilized to measure intracellular free Ca2+ concentration in murine neuroblastoma cells. The study of its hydrolysis by these cells has provided insights into the rate of formation of Fura-2 and the dynamics of its concentration within the cell, which is crucial for understanding neuronal signaling and function3.

Smooth Muscle Cell Research

Fura 2-AM has also been applied in smooth muscle cell research. The loading of the dye into cells isolated from guinea pig taenia coli has been enhanced by the use of diisopropylfluorophosphate (DFP), a cholinesterase inhibitor. This method has allowed for more efficient incorporation of Fura 2-AM, enabling simultaneous measurements of cell contraction and intracellular Ca2+ behavior4.

Plant Cell Biology

In plant cell biology, Fura 2-AM has been tested in Characean cells to measure cytosolic pCa. However, due to the challenges posed by the diffusion of the dye into the vacuole and the resulting interference with the fluorescence signal, alternative methods such as pressure injection of fura-dextran have been developed to achieve a more homogeneous distribution of the dye for accurate Ca2+ quantification5.