Fluoroskan fl microplate fluorometer

Control and mEHT treated cells were seeded in a 96-well plate, at a concentration of 10⁴ cells/well in 200 μl cell culture media. Culture media were supplemented with gemcitabine at different concentrations between 0 and 100 µM and incubated for 24, 48, and 72 h.
Resazurin is a cell-permeable redox-sensitive dye, which is reduced to resorufin by aerobic respiratory enzymes within cells. In contrast to resazurin, resorufin fluoresces when exposed to green light, thereby it is widely used to measure the viability of cells. Resazurin sodium salt (R7017 Sigma Aldrich, St. Louis, MO, United States) was diluted in PBS to make a stock solution of 0.3 mg/ml concentration which was further diluted in 1:10 when added directly into cell culture media. The measurement was done using Fluoroskan FL Microplate Fluorometer (5200110 Thermo-Fisher, Cheshire, United Kingdom) at excitation and emission wavelength of 570/590 after 2 h incubation of resazurin.

The ROS generation was determined using the fluorogenic probes H2DCFDA according to the manufacturer’s instructions. Briefly, HMC3 cells were seeded in a 96-well plate (at a density of 10,000 cells/well) and maintained in complete culture media for 24 h. Afterward, cells were washed once with HBSS and incubated for 45 min at 37 °C with 10 µM H2DCFDA dye. After incubation, the dye was removed and cells were treated for 24 h with rotenone 100 nM, control medium, or H₂O₂ used as a positive control. Cell staining was performed in HBSS. The emitted fluorescence intensity was measured using a Fluoroskan FL Microplate Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) with wavelengths of 485 nm (excitation) and 520 nm (emission).

BV2 and SH-SY5Y cells (10,000 or 50,000 cells/well, respectively) were seeded in a 96-well plate and allowed to adhere overnight. Cells were then pre-treated with either ACE or control medium for 24 h. Afterwards, they were washed once with HBSS and incubated for 45 min at 37 °C with 10 µM H2DCFDA dye. After incubation, dye was removed and cells treated for 8 h with ROT, ACE, ROT + ACE or control medium. ROT concentrations used were 5 µM for BV2 and 15 µM for SHSY-5Y cells, respectively, while ACE was always used at 10 µM for both cell lines. Cell staining and treatment were performed in HBSS and the plates were read on a plate scanner (Fluoroskan FL Microplate Fluorometer, Thermo Fisher Scientific, Waltham, MA, USA) using 485/520 nm excitation/emission wavelengths.