Cytosolic and Mitochondrial Calcium Imaging

The 661W cells were cultured on cover glass chambered slides (Nunc Lab-Tek; Thermo Fisher Scientific, Waltham, MA, USA) with the same medium described above. After treatment with HG or HG/melatonin for 24 hours, cells were directly loaded with 2 μM Fluo-4 (Thermo Fisher Scientific) and 2 μM rhodamine-2 (Rhod-2; Thermo Fisher Scientific) for 30 mins at 37°C for cytosolic and mitochondrial Ca²⁺ imaging [47 (link), 48 (link)]. After washing, new culture medium was added, and then fluorescent images were taken under identical settings, including the light intensity, exposure time, and magnification. The average fluorescent intensity per pixel for each image was quantified without any modification using the luminosity channel of the histogram function of Photoshop 6.0 (Adobe Systems, San Jose, CA, USA). A total of 8 to 11 cell images from each group were analyzed from 3 different sets of experiments [45 (link), 46 (link)].