H2dcfda

The following inhibitors and reagents were used: H2DCFDA was purchased from MedChemExpress (HY-D0940), CCK8 kit was purchased from MedChemExpress (HY-K0301), Cisplatin was purchased from Selleck (S1166), hydroxyurea was purchased from MERK (H8627) and TH5487 was purchased from MedChemExpress (HY-125276). Hoechst 33258 for nuclear staining was purchased from MERK (94403).

Cells were isolated from eye balls and prepared to single-cell suspensions. The cell activity was detected with Zombie NIR™ Fixable Viability Kit (APC-Cy7, catalog 423105, Biolegend, San Diego, CA, USA). Then, cells were stained with fluorochrome-conjugated mAbs surface markers CD11b (Bv605, catalog 101237, Biolegend) for 15 min. For detection of reactive oxygen species (ROS), cells next were incubated with H2DCFDA (FITC, catalog HY-D0940, MedChemExpress, Monmouth Junction, NJ, USA) to detect the ROS at 37 °C for 20–30 min. Finally, washing cells with cold PBS for three times before analyzing with flow cytometry (BD LSR Fortessa, BD Bioscience, San Jose, CA, USA). All data were analyzed using FlowJo (TreeStar, Ashland, OR, USA).

The intracellular reactive oxygen species (ROS) level was monitored using the ROS sensitive probe (2′,7′-dichlorodihydrofluorescein diacetate, H₂DCFDA, Invitrogen, D-399). To make a 10 mM stock solution, H₂DCFDA was diluted in Dimethyl sulfoxide (DMSO, MedChemExpress, Monmouth Junction, NJ, USA, HY-15534) and then further diluted before use. Adherent cells (Human Adipose Stem Cells, ADSC) in a flow chamber were stained for 30 min in the dark at room temperature with a 5 µM staining solution in PBS. Then, using an 830 nm infrared diode laser, the control and PBM-exposed cells were examined at various flux intensities. The H₂DCFDA staining method was the same as the H₂DCFDA staining method. The laser irradiation protocol is followed as in Table S2. At fluences of 2.5, 5, and 10 J/cm²), intracellular ROS was expressed as a ratio (R₂/R₁) before (R₁) and after (R₂) 30 min of PBM.

The H₂DCFDA staining assay was performed according to a previously reported method with slight modifications [32 (link)]. The excised leaves were stained with 10 μM H₂DCFDA (MedChemExpress, Monmouth Junction, NJ, USA) in 10 mM PBS buffer in the dark for 30 min. Images were captured under an Olympus FV3000 confocal laser scanning microscope (Olympus Corp., Tokyo, Japan) with a 488 nm filter. ROS signals were visualized in the range of 501–550 nm, and chlorophyll autofluorescence was detected in the range of 640–735 nm.

Fluorescence-based measurements of mitochondrial membrane potential and intracellular ROS generation were performed as previously described 50 (link). In brief, following treatment, hepatocytes were incubated with the potentiometric JC-1 sensor (Mitochondrial Membrane Potential Detection Kit, #30001, Biotium). For ROS measurements, fresh liver sections or primary hepatocytes were incubated with the cellular ROS indicator DHE (#PD-MY 003, MedChemExpress), cytoplasmic ROS indicator H2DCFDA (#HY-D0940, MedChemExpress), and mitochondrial ROS indicator MitoSOX RED (HY-D1055, MedChemExpress) based on methods provided by manufacturers 51 (link). Fluorescence images were acquired with same laser output and gain for control and treated samples on a Zeiss 880 confocal microscope using a 100X oil objective 52 (link).