Metafluor imaging and analysis software

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.

At 18–24 h after nucleofection with GCaMP3-ML1, podocytes were used for experiments [26 (link)]. The fluorescence intensity at 470 nm excitation (F470) was recorded with a Nikon Diaphoto TMD Inverted Fluorescence Microscope. Metafluor imaging and analysis software were used to acquire, digitize and store the images for offline processing and statistical analysis (Universal Imaging, Bedford Hills, NY, USA). Lysosomal Ca²⁺ release was measured under a ‘low’ external Ca²⁺ solution, which contained 145 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 10 mM glucose, 1 mM EGTA and 20 mM HEPES (pH 7.4). The response to ML-SA1 (20 μM), a TRPML channel agonist and activator of lysosomal Ca²⁺ release, was determined. The Ca²⁺ ionophore, ionomycin (1 μM) was used as a positive control in the podocytes. After GCaMP3 Ca²⁺ imaging, we compared the peak value of ΔF/F₀ ((intracellular F470 intensity at n second−intracellular F470 intensity at 0 s)/intracellular F470 intensity at 0 s) between additions of ML-SA1 and ionomycin to solution.