Cells were plated on µ-Slide 8 Well ibiTreat chamber slide (IBIDI) and grown for an additional 24 h. Cells were then left untreated or treated with 200 µM sodium arsenite diluted in complete media or 20 µM clotrimazole diluted in serum-free medium for 30 min then viewed with a Supercontinuum Confocal Leica TCS SP5 X, equipped with a pulsed white light laser, 405 nm violet diode laser, a Leica HCX PL Apo 63x/1.40 oil objective and a heated chamber set on 37°C. Live cell images were recorded sequentially with the following settings to avoid saturated signals. Pinhole was set to 2 airy. First: excitation at 485 nm with 25% intensity of the white laser light, PMT1 (photomultiplier tube) detector was set to gain 900 V and offset −1.0 V, emission window was set from 505 to 515 nm. Second: excitation at 405 nm of the violet diode laser with 25% intensity, PMT1 detector was set to gain 900 V and offset −1.0 V, emission window was set from 505–515 nm. Quantification was performed on four images with 3–4 cells and multiple SGs per cell, which totalled around 40 or more SGs per experiment. The area of the region of interest (ROI) was set equal in SGs and in adjacent cytoplasm and analysed. Then, the mean pixel values of channel 485 nm and 405 nm were measured, and the 405/485 nm ratio was calculated and plotted [58 (link)].
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