Intracellular Free Calcium Measurement Using Confocal Imaging
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4. After the loading period, carefully recover or the coverslip and wash the cells three times with Tyrode-BSS buffer. 5. Leave the loaded cells for an additional 15 min period to ensure complete hydrolysis of acetoxymethyl ester groups. 3.2 Acquisition of Intracellular Calcium Whole-Cell Images
1. After cleaning the objective lens, a drop of nonfluorescent oil is applied and then the chamber containing the loaded cells is placed in contact with the objective lens. Using the lowest possible intensity of the mercury lamp of the microscope, we choose a cell that presents a stable fluorescence level and that does not have a flat appearance. This should be done without exposing the chosen cell for a long period of time to mercury lamp light in order to avoid photobleaching. 2. The mercury lamp is turned off and the shutter of the microscope is set in the position allowing the passage of the laser excitation waves. 3. At first, a continuous section scanning is done in order to estimate the basal level of cytosolic and nuclear calcium. The cytosolic level should be between 50 and 100 nM, and the nuclear level should be all the time higher than that of the cytosol (near 300 nM) according to the calibration curve already determined for the calcium fluorescent dye. This permits all the cells used to have nearly the same starting normal calcium. During this process, the focus should be adjusted with the fine adjustment knob. 4. When the continuous sectioning is terminated, we proceed to the determination of the thickness of the cell by performing a vertical scan which allows the determination of the starting section (just above the cell) and the number of sections needed to scan the whole cell. The step size should be kept at the minimum value. 5. Serial Z-axis optical scans (section series) taken for the intracellular calcium of the cell are captured by a photodetector, digitalized, and saved (Fig. 1a). The captured section series can then be presented either as 2D or real quantitative 3D reconstructions using various angles of rotation and inclination as well as a variety of cutting planes. These real quantitative 3D reconstructions of cells (Fig. 1b, c) are then used for the measurement of basal level fluorescence intensity and/or the cellular response after the addition of different agents [1–5]. 6. At the end of each experiment, the nucleus is stained with 100 nM of the live nucleic acid stain Syto-11 (Fig. 1c) (Molecular Probes, OR, USA) [1–5]. Serial Z-axis optical scans are taken after development of the stain (3–5 min) while maintaining positioning, number of sections, and step size identical to those used throughout the experiment. Nuclear labeling is
