Imaging Ca2+ Dynamics in Cells

Changes in [Ca²⁺]_i were monitored in cells labeled with the Ca²⁺-sensitive fluorescent indicator, Calcium Green-1(Ex_{480 nm} and Em_{535 nm}) as previously described^{18 (link)}. For dye loading, the cells were incubated with 1 µM Calcium Green-1 for 45 min at 37 °C in a balanced salt solution (BSS) containing 0.1% bovine serum albumin (BSA) and (in mM): 145 NaCl, 5 KCl, 1.2 NaH₂PO₄, 2 CaCl₂, 1.3 MgCl₂, 10 glucose, and 15 HEPES, pH 7.4. For fluorescence monitoring, cells were washed twice with dye-free BSS lacking BSA and placed on the stage of a Nikon TE2000 epifluorescence microscope equipped with a 100X objective. For Ca²⁺ free experiments cells were bathed in Ca²⁺-free BSS Ca²⁺ containing 1 mM EGTA. Fluorescence images of the cells were captured before, during and after application of a stimulus by an iXonEM + DU-897 EMCCD camera (Andor Technology Ltd., Belfast, UK) using the open source microscopy software Micro-Manager (version 1.4, Vale Lab, UCSF, San Francisco, CA). The exposure time of the camera was set to 100 ms and images were captured at a rate of 7.5 Hz. Bright field images were obtained at the start and end of an experiment, and all experiments were carried out at ambient room temperature.