Cellrox green

To analyse oxidative stress, cells were incubated with the indicated drugs for 2 h before CellROX Green reagent (Molecular Probes, ThermoFisher) was added to the medium at a final concentration of 5 μM for 30 min at 37 °C. Cells were then washed three times with PBS and placed under confinement for 2 h. Cells were finally fixed for 20 min in 4% paraformaldehyde and washed twice in PBS. CellROX Green fluorescence was then measured (Ex. 488/Em. 520) by confocal microscopy using identical settings (Zeiss LSM 780 NLO, Zeiss, Germany). Experiments were performed in triplicate.

A cross-sectional area of each of the isolated single PV cardiomyocytes was imaged using a confocal laser scan microscope (Zeiss LSM 510), and the acquired images were processed using ImageJ measurement tools. We used CellROX Green (Life Technologies, Grand Island, NY, USA) to assess cytosolic ROS production in the freshly isolated control and MIF-treated PV cardiomyocytes at 1 Hz pacing. The measurements were performed using a laser-scanning confocal microscope (Zeiss LSM 510) and an inverted microscope (Axiovert 100) with a 63 × 1.25 numerical aperture oil immersion objective, as described previously.^{7 (link)} Freshly isolated PV cardiomyocytes were maintained in normal Tyrode’s solution (NaCl, 137 mM; KCl, 5.4 mM; CaCl₂, 1.8 mM; MgCl₂, 0.5 mM; HEPES, 10 mM) with 10 µM CellROX Green fluorescent dye. The CellROX Green dye was excited at 488 nm, and fluorescent signals were acquired at wavelengths of >505 nm in the XY mode of the confocal system. The acquired fluorescent images were analysed using Image-Pro Plus 6.0 and Sigma Plot 12 software, as described previously.⁹

CellROX^®Green (Invitrogen, Carlsbad, CA, USA) staining was used to detect mtROS levels in GP-treated worms. The worms were incubated in NGM agar plates containing 5 µM CellROX^®Green dye for 1 h at 20 °C, and the CellROX^®Green signal was observed. Dissected gonads and oocytes were also analyzed by dissecting worms in 5 µM CellROX^®Green dye on a poly L-lysine-coated slide and incubated in a wet chamber for 30 min at 20 °C. The CellROX^®Green signal was observed under a fluorescence microscope (Zeiss Axioscope, Oberkochen, Germany), and its intensity was quantified using ImageJ software (v.1.53e software, National Institute of Health, Bethesda, MD, USA). NAC (Sigma-Aldrich, St. Louis, MO, USA) was used to examine the antioxidant effect. Worms fed with GP were treated with the antioxidant NAC. Synchronized L4-stage worms were placed on NGM plates containing 0 or 3 µM GP for 48 h at 20 °C, and then the worms were transferred to 0 or 5 mM NAC-treated NGM plates containing 0 or 3 µM GP for 24 h at 20 °C.