Gemini em microplate reader

The ARPE-19 cells were harvested and seeded into the 96-well plates at the density of 2 × 10³ per well. The high-glucose (50mM) were then incubated with the cells for 0h, 12h, 24h and 36h, respectively. The commercial CCK-8 kit (AbMole, USA) was employed to measure cell proliferation according to the manufacturer’s protocol. Briefly, 10 μl of CCK-8 solution was added into each well for 4 h. After that, the plates were gently mixed and the Gemini EM microplate reader (Molecular Devices, USA) was used to measure the optical density (OD) values at the absorbance of 450 nm. The OD values were used to reflect the proliferation abilities of ARPE-19 cells.

Cells were washed 1× in PBS and resuspended in 1 mL PBS. Next, 2 μL monochlorobimane solution (2.2 mg monochlorobimane in 194.12 μL acetonitrile) was added to each sample. Samples were vortexed and incubated at 37 °C for 15 min. The reaction was halted by adding 50 μL trichloroacetic acid and vortexing. Samples were spun for 5 min at 10,000 rpm, and 1 mL supernatant was added to a glass tube containing 1 mL dichloromethane. Glass tubes were vortexed and centrifuged for 2 min at 3,500 rpm. For each sample, 200 μL of the top aqueous layer was plated in duplicate wells in a white 96-well plate. Fluorescence was read on a Gemini EM Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation of 360 nM and an emission of 460 nM. Glutathione concentrations were determined by comparing samples to a standard curve composed of varying concentrations of glutathione ethyl ester dissolved in PBS.

After UVB-irradiation and PRE treatment (0.5–5 μg/mL), the HS68 cells were stained with 40 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich) for 30 min. ROS production was analyzed by Gemini EM Microplate Reader (Molecular Devices) with excitation and emission wavelengths of 488 nm and 520 nm, respectively.

The GENMED oxidative stress reactive oxygen species (ROS) primary fluorescence assay kit for living tissue (GMS10016.4, GENMED Scientifics Inc., MA, USA) was chosen to detect ROS. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) is a stain that can penetrate through the cell membrane freely. Green fluorescence is produced once DCFH-DA is oxidized by hydrogen peroxide, peroxide groups, peroxynitrite, etc. Briefly, the hippocampus was freshly harvested 24 h after stereotaxic injection and cold GENMED diluent (10 mg/100 μL) was quickly added. Tissues was homogenized with a DOUNCE homogenizer on ice. The protein in the hippocampus was determined by the GENMED Bradford protein assay kit (GMS30030.1, GENMED Scientifics Inc., MA, USA). Fifty microliters of the supernatant was mixed with 50 μL catalyst in a well of 96-well plates and incubated at room temperature for 5 min. Subsequently, GENMED staining working solution containing DCFH-DA (100 μL) was added to each well and reacted for 20 min in the dark. Fluorescence at 490 nm excitation and 520 nm emission was read on a fluorescence microplate reader (Gemini EM Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) and observed under an Olympus BX5 fluorescence microscope imaging system (Olympus America, Melville, NY, USA).