MPTQ-mediated cell death in neuro 2a and SH-SY5Y was studied using Live-Dead assay kit (Invitrogen, USA). The kit has two fluorescent dyes, calcein-AM and ethidium homodimer. In principle, calcein-AM can enter any cells but labels only live cells. It is converted by cellular cytoplasmic esterases to a highly green fluorescent calcein. Ethidium homodimer is excluded by live cells with intact membrane but enters dead cell with broken membrane to stain their nuclei red. Therefore, live cells fluoresce green where as dead cells fluoresce red. Neuro 2a and SH-SY5Y cells were seeded at 20,000 cells/well and 100,000 cells/well respectively in 24-well tissue culture plates. After 48 hrs, neuro 2a cells were treated with 2.5, 5, 10, 20 and 30 µM of MPTQ whereas SH-SY5Y cells were treated with 90 µM of MPTQ in their respective cell culture medium. Control cells were treated with equal volume of DMSO. Live dead assay was performed 24 hrs post treatment in neuro 2a cells but after 4days in SH-SY5Y cells. Live-Dead reagent was diluted either in serum free DMEM or PBS (1X) to 2X concentration and added directly to the cell culture media at 1∶1 ratio followed by a gentle but thorough mixing. Cells were incubated in dark for 30 minutes at room temperature. After incubation, 3–4 random images were captured per well using an inverted microscope (Nikon Ti Eclipse, Japan) supported by quantis monochromatic cooled CCD camera, MetaMorph software and using FITC (for calcein) and Texas Red (for ethidium homodimer) filter cubes. Number of live (green) and dead (red) cells were counted using the multi-wavelength cell scoring module of MetaMorph software. For time-dependent cytotoxicity of MPTQ on neuroblastoma cells, neuro 2a cells were treated with 30 µM of MPTQ for 24, 48, 72 and 96 hrs and Live-Dead assay was performed as described above. Full paper
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