Measuring Intracellular Calcium Levels with Fura-2

A fluorescence image analysis system was used to determine [Ca²⁺]_i in CAMs with fura-2 acetoxymethyl ester (fura-2) as an indicator as previously described [14 (link)], Having been loaded with 10 μM fura-2 at room temperature for 30 min, the cells were washed three times with Ca²⁺-free Hank’s buffer. The ratio of fura-2 emissions, when excited at the wavelengths of 340 and 380 nm, was recorded with a digital camera (Nikon Diaphoto TMD Inverted Microscope). Metafluor imaging and analysis software were used to acquire, digitize, and store the images for off-line processing and statistical analysis (Universal Imaging). The fluorescence ratio of excitation at 340 nm to that at 380 nm (F340/F380) was determined after background subtraction, and [Ca²⁺]_i was calculated by using following equation: [Ca²⁺]_i = K_dβ[(R − R_min)/(R_max − R)], where K_d for the fura-2-Ca²⁺ complex is 224 nM; R is the fluorescence ratio (F₃₄₀/F₃₈₀); R_max and R_min are the maximal and minimal fluorescence ratios measured by addition of 10 μM of Ca²⁺ ionophore ionomycin to Ca²⁺-replete solution (2.5 mM CaCl₂) and Ca²⁺-free solution (5 mM EGTA), respectively; and β is the fluorescence ratio at 380-nm excitation determined at R_min and R_max, respectively.