Ts eclipse inverted microscope

SH-SY5Y cells (2 × 105 cells/well) were seeded in 24 well tissue culture plates and further treated with Oxa and Mel in their respective medium. Cell images were captured from 6 random fields at 20x magnification using NIS elements software provided with Nikon Ts Eclipse inverted microscope.

Imaging or fluorometric measurement of mitochondrial membrane potential in intact SH-SY5Y cells was achieved by using membrane potential dependent fluorescent TMRE (Sigma, USA) probe [22 (link)]. This cationic, lipophilic dye enters cells in the form of an ester. It is then hydrolyzed to produce tetramethylrhodamine, a product that accumulates within the mitochondria due to a high membrane potential. Cells were seeded at a density of 5 × 105 in two well chamber slide for 24 h in 5% CO2 incubator at 37 0C. Thereafter, cells were pre- treated with 10 μM Mel for 1 h and later with or without 50 μM Oxa for 24 h. Cells were then washed twice with PBS and further stained or incubated with TMRE (100 nM) for 15 min at 37 0C. The stained cells were visualized with excitation at 540 nm and emission at 590 nm by using NIS elements software provided with Nikon Ts Eclipse inverted microscope. Five to six images were captured at 20x magnification. Image scales were burned on to each picture using the same software. Maximum projections were generated from focus planes and images were displayed with equal pixel intensity. The TMRE fluorescence integrated density was analyzed with Image J software (1.50 version, NIH, USA).

Superoxide anion production was detected using the dihydroethidium (DHE) probe. 5 × 105 cells/well were seeded on 6-well tissue culture plates and allowed to adhere for 24 h in a 5% CO2 incubator at 37 0C. Thereafter, cells were treated with Oxa and Mel in their respective medium. Further, cells were stained or incubated with DHE solution (2 μg/ml) for 20 min. DHE dye incorporated into the cells and subsequently oxidized to the fluorescent ethidium cation by superoxide anion, allowing the cation to bind nuclear DNA with an extensive enhancement of fluorescence at 530 nm when excited at 485 nm [23 (link)]. The fluorescence signal was detected as proportional to superoxide production. The DHE fluorescence integrated density was analyzed with Image J software (1.50 version, NIH, USA). The 3–4 images were captured using NIS elements software provided by the Nikon Ts Eclipse inverted microscope at 20x magnification. Maximum projections were generated from focus planes and images were displayed with equal pixel intensity. Results were expressed as percentage of DHE stained positive cells.