Cellrox green probe

Reactive oxygen species (ROS) variations were monitored through the microplate reader and the CellROX^® green probe (Thermo Fisher Scientific, Waltham, MA, USA). BAE-1 were seeded (3.3 × 10⁴ cells/200 μL/well) on black clear bottom 96-well plates (Greiner Bio-One, Kremsmünster, Austria) and incubated at 37 °C for 24 h. Following incubation, cells were treated according to experimental conditions: TMAO 100 μM for 1 h, MEN 100 μM for 1 h, TMAO 100 μM + MEN 100 μM for 1 h, for acute stimulation tests, and TMAO 100 μM for 24 h, MEN 100 μM for 1 h, TMAO 100 μM for 24 h + MEN 100 μM for 1 h, for prolonged stimulation tests. CellROX^® green (5 μM) was added in each well 30 min before the end of the treatment and cells were then washed twice with PBS containing Ca²⁺ and Mg²⁺ to avoid cell loss. Fluorescence intensity variations were measured at 485 nm through the FilterMax F5TM Multi-Mode microplate reader. ROS production was expressed as mean fluorescence compared to the control of three independent experiments.

For fluorescence microscopy comparison against CellROX Green probe (Invitrogen, USA, Catalogue number: C10444), the DH5α/pJ404-IscR-C cells grown as above were divided into three parallel cultures and incubated for an additional 2 h at (i) 37 °C (control), (ii) 23 °C (cold stress), and (iii) 37 °C in the presence of 2 mmol L⁻¹ H₂O₂ supplied at one-hour intervals. The cultures were then supplemented with 5 μM of the CellROX Green, followed by an additional 30 min incubation in the dark at 37 °C. All samples were then subjected to simultaneous analysis of red fluorescence (E2-Crimson expression from pJ404-IscR-C) and green fluorescence (CellROX Green) using confocal fluorescence microscopy.

The oxidative stress generated by optical and electrical stimulation was measured by a fluorogenic CellROX^® green probe (Invitrogen; Carlsbad, CA). Four different groups of NRVMs were seeded on 24-well plates. One group was photostimulated for 3 min using 1 Hz light pulse. The second group was electrically stimulated using the same frequency. Finally, a negative (untreated cells) and positive [treated with 200 μM menadione for 1 h (Sigma-Aldrich, St Louis, MO, USA] control were performed with the third and fourth group. Cells, after stimulation and menadione treatment, were incubated in 5 μM CellROX^® green and 20 ng ml⁻¹ Hoechst stain (ThermoFisher Scientific, Waltham, MA, USA) for 30 min. Samples were rinsed with PBS, fixed in 4% PFA, and imaged immediately using an EVOS M7000 Imaging System (ThermoFisher Scientific, Waltham, MA, USA). CellROX^® intensity values were normalized to the number of Hoechst-positive cells and corrected for background fluorescent intensity of cells and NRVMs without CellROX^®.