H2dcfda

Cells were seeded in 6-well plates, exposed to DMSO or indicated compounds for 72 h, and harvested by trypsinization. For total cell ROS analysis, cells were stained with 0.1 μM H₂DCFDA (Biotium, Fremont, CA, USA) at 37 °C for 20 min. For mitochondrial ROS analysis, cells were stained with 5 μM MitoSOX Red (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 10 min. After washing with PBS three times, cells were subjected to ROS detection via flow cytometry with CellQuest software according to the manufacturer's instructions (Becton Dickinson, Mountain View, CA, USA), as previously described [47 (link)].

ROS activity in the animal body after irradiation was identified using H2DCFDA (2,7-dichloro-dihydrofluorescein-diacetate-acetyl) or CellROX^® Green Reagent. This dye is a well-known fluorescent intracellular sensor of reactive oxygen species [35 (link)]. Planarians were put in a solution of 10 μM H2DCFDA (Biotium, Fremont, CA, USA) and incubated for 1 h in darkness. Next, the animals were treated with Tameron, washed twice with water, and irradiated using an X-ray device or incubated in menadione solution. The positive control group was obtained by pre-incubation of animals for 30 min in 100 μM H₂O₂ (Sigma, Saint Louis, MO, USA) or CellROX^® Green Reagent (C10444, Thermo, Carlsbad, CA, USA). Then, the planarians were anesthetized for 5–10 min in a 0.1% solution of chloretone (Sigma, Saint Louis, MO, USA) [36 (link)] and photographed with an Axio Scope A1 fluorescence microscope (Carl Zeiss, Jena, Germany) (Ex/Em = 492–495/517–527 nm). In the images obtained using the ImageJ program (National Institute of Health, Bethesda, MD, USA), the total fluorescence intensity of the animal body was evaluated. The measurement results were averaged over 15 animals.

Cytosolic ROS production was measured by using cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Biotium). Upon cleavage of the acetate groups by intracellular esterase and oxidation, the nonfluorescent H2DCF-DA is converted to the highly fluorescent DCF. Briefly, cells were treated with various agents for the desired length of time and then incubated with HBSS (GIBCO®) containing 10 μM H2DCF-DA for 30 minutes at 37°C/5% CO₂. Cells (>5000) were collected using ACCUTASE™ (STEMCELL™), washed, and resuspended in PBS prior to analysis using a ACEA NovoCyte™ flow cytometer (Agilent Technologies, No. 205, Zhaohui Road, Hangzhou, Zhejiang, China). Cytometer settings were optimized for green (FITC-H) fluorescence, and data were analyzed with the NovoExpress 1.4.1 Software (Agilent Technologies, No. 205, Zhaohui Road, Hangzhou, Zhejiang, China).

For cell-cycle analysis, cells were seeded at 1 × 10⁵~2 × 10⁵ cells per well on 6-well plates and incubated overnight. The following day, cells were treated with DMSO (0.05%) or various concentrations of compounds for the indicated time without changing the media. After the treatment, cells were harvested by trypsinization, washed with 1 mL of phosphate-buffered saline, and fixed in ice-cold 75% ethanol at −20 °C overnight. Subsequently, the cells were stained with propidium iodide (80 µg/mL) containing 0.1% Triton-X 100 and 100 µg/mL of RNaseA. DNA content was analyzed using FACScan and CellQuest software (Becton Dickinson, Mountain View, CA, USA). For ROS analysis, cells were stained with 0.1 μmol/L H₂DCFDA (Biotium, Fremont, CA, USA) at 37 °C for 20 min. After washing with PBS three times, the cells were subjected to ROS detection via FACScan and CellQuest Pro 4.0.2 software.

Cell culture media and solutions were obtained from Biological Industries (Kibbutz Beit Ha’emek, Israel). Fluorescent mitochondrial dyes were purchased from Invitrogen (Carlsbad, CA, USA), H₂DCFDA from Biotium (Hayward, CA, USA), and the ATPlite^TM was from Perkin Elmer (Waltham, MA, USA). Other reagents were purchased from Sigma-Aldrich (Rehovot, Israel) at the highest purity available.