Since calcium plays a critical role in cell function it is important to be able to determine the extent to which chemical interactions affect the redistribution of this divalent cation. There are a number of dyes useful for these measurements such as Indo-1, fura-2 and fluo-3. Indo-1 is an excellent dye for flow cytometric measurement of free intracellular calcium but it does require a UV light source. This dye has the ability to undergo a fluorescent wavelength emission shift when bound to calcium. Indo-1 is introduced to the cells as an acetoxymethyl ester which undergoes enzymatic hydrolysis in cells to yield free dye.
Flow cytometry has proved to be a valuable resource in the evaluation of the role of calcium in neutrophil function. A major spectral change can be measured when indicators of Ca2+ penetrate cells and are excited at 357 nm (ultra-violet excitation). Cells are loaded with Indo-1 (final concentration 3 um) for 15 min at 37oC and then immediately run on the flow cytometer to obtain fluorescence histograms at two emission wavelengths; 395 nm (bound Ca2+) and 525 nm (non-bound calcium). The Ca2+ concentration of cells can be determined independently of dye concentration by evaluating the ratios of the two fluorescent emissions. Thus a high 395/525 nm ratio would indicate bound Ca2+. Ionomycin is used as a positive control for measurement of calcium flux. Ionomycin (3-5 mM) will cause an increase in the BOUND (long wavelength) fluorescence signal (i.e. increase in BOUND [Ca2+] inside the cell).
