Tilapia NIL cells were cultured on poly-D-lysine precoated coverslips (~5 × 105 cells/ml/coverslip) for single-cell Ca2+ imaging as described previously (28). Briefly, tilapia NIL cells were preloaded with the Ca2+-sensitive dye Fura-2-AM (5 µM, Molecular Probes) using a microspectrophotometry fluorescence-ratio setup equipped with a perfusion system at room temperature for 40 min. After that, the ratiometric measurement of intracellular Ca2+ level ([Ca2+]i) was measured with excitation wavelengths at 340 and 380 nm using a PTI Epifluorescence Ca2+ Imaging System (Photon Technology International, Birmingham, NJ) Ca2+ data were expressed as a ratio of fluorescence signals with excitation at 340 and 380 nm, respectively (as “F340/380 Ratio”).

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