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Text from PDF Page: 003Cells 2021, 10, 2635 3 of 24 of DMSO was maintained below 0.1%, since our previous studies revealed that this con- centration does not cause cellular damage. 2.3. Cell Viability Assay and Morphology Examination In the determination of the cytoprotective effect of catalpol on ARPE-19 cells, cell viability was measured using MTT assay. The cells were plated in 96-well plates at a den- sity of 5 × 103 cells/well overnight. After treatment with varying concentrations of catalpol (10–40 μM) for 24 h, H2O2 (400 μM) was added and treatment was continued for another 6 h. Untreated cells served as control. After incubation, the cells were treated with 100 μL MTT working solution (0.5 mg/mL in new culture medium) for 4 h at 37 °C. Thereafter, 150 μL DMSO was added to each well to dissolve the water-insoluble formazan crystals formed. Cell viability was evaluated by measuring absorbance of the formazan solutions at 570 nm using a microplate reader (Thermo, Multiskan, GO, United States). Morpholog- ical changes in ARPE-19 cells were observed and photographed under an inverted Olym- pus IX71 microscope. 2.4. LDH Cytotoxicity Assay The release of lactate dehydrogenase (LDH) was determined using a LDH cytotoxi- city assay kit. The ARPE-19 cells were inoculated into 96-well plates at a density of 1.5 × 104 cells/well and incubated as indicated in Section 2.3. At the end of treatment, the cells were centrifuged at 1000 rpm for 5 min to obtain supernatants. According to the manu- facturer’s instructions, 100 μL of each supernatant was used to determine the content of LDH. The absorbance of the sample was determined on a Thermo microplate reader (Mul- tiskan, GO, United States) at 490 nm with dual wavelength, using 600 nm as reference wavelength. 2.5. TUNEL Staining One of the recognized marker events of apoptosis is DNA fragmentation. The specific DNA fragments can be measured using a terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay. The ARPE-19 cells were seeded on coverslips at a density of 2 × 105 cells/well and treated as indicated in Section 2.3. Thereafter, the cells were fixed with 4% (w/v) paraformaldehyde. Then, apoptosis was determined using TUNEL assay kit according to the manufacturer’s instructions. Apoptotic cells were iden- tified and photographed using a florescence microscope (BX51-DSU; Olympus, Tokyo, Japan) at × 200 magnification. 2.6. Apoptosis Assay The ARPE-19 cells were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured overnight. The cells were then incubated with either catalpol (10-40 μM) for 24 h, or NAC (10 mM) for 2 h before treatment with 400 μM H2O2 for 6 h. In brief, the cells were collected and stained in 300 μL of binding buffer containing 5 μL Annexin V–fluo- rescein isothiocyanate (FITC) and 10 μL propidium iodide (PI) for 25 min at room tem- perature in the dark. The stained cells were subjected to apoptosis analysis using a FACSCanto II flow cytometer (BD Biosciences, New Jersey, USA). 2.7. Measurement of ROS Levels The levels of intracellular ROS were determined with 2,7-dichlorofluorescin diacetate (DCFH-DA) staining assay. The ARPE-19 cells were plated in 6-well plates at a density of 2 × 105 cells/well and incubated as stated in Section 2.3. Thereafter, the cells were harvested and incubated with 10 mM DCFH-DA working solution using ROS assay kit at 37 °C for 30 min in the dark. The cells were washed and re-suspended in PBS, followed by flow cytometric analysis using flow cytometer to determine the percentage of fluorescence- positive cells.
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