DETERMINATION OF IN-SITU DISSOCIATION-CONSTANT FOR FURA-2 AND QUANTITATION OF BACKGROUND FLUORESCENCE IN ASTROCYTE CELL-LINE U373-MG

Accurate estimates of cytosolic free Ca2+ with fluorescence indicators are dependent on the determination of the in situ dissociation consta nts (k(d)) of the intracellular dyes and the correction for background fluorescence. The in situ dissociation constant for Ca2+ and indicato r dye Fura-2/AM varies significantly from the in vitro published value s due to differences in ionic strength, pH, viscosity and Ca2+ bufferi ng by intracellular lipids and proteins. During the course of a measur ement, background fluorescence changes may occur as the result of endo genous fluorescent compounds and compartmentalized Fura-2 indicator. T he in situ dissociation constant value was determined for human astroc yte cell line U373-MG by creating several known intracellular Ca2+ con centrations while measuring total fluorescence and background fluoresc ence values for each. The background fluorescence was not constant, ra ther it demonstrated a linear relationship with the free cytosolic Ca2 + concentrations and total fluorescence intensities. The Ca2+ dependen t and total fluorescence dependent background was expressed as a linea r equation and subtracted appropriately from the total intensity measu rements. The in situ dissociation constant was determined to be 3-fold greater than in vitro measurements after the background was corrected . The experimentally determined standard linear equations for backgrou nd quantitation and the in situ dissociation constant for this line pr oduce accurate cytosolic free Ca2+ estimates.