Dcfh da

Trabecular were isolated from femur and ground with liquid nitrogen. The homogenate in PBS was centrifuged to remove debris and the supernatants were incubated in 10 μM dichorodihydro fuorescein diacetate (DCFH-DA; Molecular Probes, GenePharma, Suzhou, China) for 10 min at room temperature. DCFH-DA oxidation into 2′,7′-dichlorofluorescein was measured using a spectrofluorometer (Bio-TEK, Vermont, USA, excitation 485 nm and emission 525 nm). Date were expressed as value of optical density (OD).

The fluorescent probe 2’7’-dichlorofluorescein-diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO) was used to measure ROS production in Bge cells following a method described previously with hemocytes [33 ]. Bge cells (~1.5 x 10⁵) in suspension were washed 3X with CBSS before incubation in CBSS (control), CBSS containing either 30 μg/mL LTP, 1 mM ZnCl₂ or 30 μg/mL LTP + 1 mM ZnCl₂ for 1 hr at 26°C. After treatment, cells were washed 3X with CBSS and centrifuged at 1000 rpm for 10 min. The final cell pellets were then re-suspended in 150 μL of CBSS containing 10 μM DCFH-DA, and distributed in three wells of a 96-well black-walled plate (BD Falcon). The oxidation of DCFH-DA to fluorescent 2’7’-dichlorofluorescein (DCF) was measured in triplicate at 10 min intervals for up to 60 min using a Bio-Tek Synergy fluorescence plate reader (Winooski, VT) with excitation and emission wavelengths of 485 ± 20 and 528 ± 20, respectively. Data analysis was conducted with Origin software (Microcal, Northhampton, MA, USA). Five independent replicates of each experiment were conducted, with the raw data presented as mean ± SEM, and ratios of means of treated groups to controls presented separately.

~1 × 10⁵ LCLs (both LCL#1 and LCL#89) either left untreated (DMSO control) or treated with 0.5 μM MG132 in the presence and absence of ROS (Reactive Oxygen Species) scavenger 1 mM N-Acetyl-L-cysteine (NAC) for 24 h were harvested, suspended in PBS in a 96-well plate (Corning Inc., NY, USA). The fluorescent probe DCFH-DA (Sigma-Aldrich Corp. St. Louis, MO, USA) was used to detect intracellular ROS levels. After incubation with 20μM DCFH-DA for 30 min at 37°C and the fluorescence was measured by Synergy H1 Multimode Microplate Reader (BioTek Instruments, Inc., VT, USA) using the blue filter (485 nm) for excitation and the green filter (528 nm) for emission. Experiments were performed in triplicate and were independently repeated two times.

The cell permeable reagent 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma–Aldrich) was used to detect post-irradiation ROS production in both R28 cells and mouse retinas, and MitoSOX Red (Yeasen) was used to detect mitochondrial superoxide, of which Hoechst (Apexbio, Houston, USA) was applied to indicate nucleus of living cells. Cells in 96-well plates were washed twice with low glucose DMEM and the media were then replaced with DMEM containing 25 µM DCFH-DA. Cells were then incubated in the dark for 30 min, after which they were washed twice and resuspended in DMEM. The mouse eyes were enucleated after irradiation; retinas were then immediately frozen and homogenized in low glucose DMEM using a tissue grinder (KZ-II, Wuhan, China). The tissue homogenates were incubated with DCFH-DA (50 µM) at 37 °C in the dark for 30 min. Samples were then centrifuged at 3000 rpm for 5 min at 4 °C. Finally, the resulting pellets were washed twice with cold DMEM and then resuspended in low glucose DMEM. DCFH-DA fluorescence was measured at 488 nm of exciting light and 525 nm of emitting light using a FLUOstar OPTIMA Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). As for MitoSOX Red staining, cells were incubated with 2 µM fluorochrome for 10 min, and then washed with PBS and examined using a fluorescence microscope (Leica, Wetzlar, Germany).