*4.3. Cytotoxicity Evaluation*

The MTT assay and LDH release assay were employed to measure cell viability and cell membrane integrity, respectively, according to a previous study [23]. Briefly, RPE cells were plated in 96-well plates at the concentration of 5 × 10<sup>5</sup> cells/mL for incubation 48 h. The cells were then treated with serum-free F12 medium containing 25.0 μmol/L DHA and different concentrations of fucoxanthin or lutein. After 24 h incubation, the cell supernatant was collected for LDH analysis. Subsequently, 150 μL of serum-free F12 medium containing 0.50 mg/mL MTT was added into each plate well and incubated for 4 h. The medium was then replaced with dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm. The LDH activity in the cell supernatant was determined using a commercial LDH kit, according to the manufacturer's instructions.

#### *4.4. Construction of Differentiated RPE Cell Monolayer*

The differentiated RPE cell monolayer was cultured as described in previous studies [39,40]. RPE cells at passage 4 were seeded onto transwell inserts with polyester membranes (6.5 mm diameter, 0.4 μm pores) from Corning Inc. (Corning, NY, USA), at a seeding density of 1 × 10<sup>4</sup> cells per well (about 30,000 cells/cm2). In the apical and basolateral chambers, 200.0 and 600.0 μL of serum-free F12 media were added, respectively. The cultures were supplied with 5% CO2 in a humidified incubator (37 ◦C). Transepithelial electrical resistance (TER) was used to evaluate the differentiation degree of the RPE cell monolayer (Figure 6). It was deemed to be usable for subsequent light damage experiments when the differentiated RPE cell monolayer was cultured for 4 weeks, and the net TERs were ≥25.0 Ohm·cm<sup>2</sup> [39].

#### *4.5. Visible Light-Induced RPE Cell Injury In Vitro*

Visible light- and lipid-induced RPE cell damage was performed based on our previous reports [41], with some modifications. Subsequent to the formation of the differentiated RPE cell monolayer, serum-free F12 medium containing 25.0 μmol/L of DHA was added into the apical chamber, whereafter the RPE cell monolayer was subjected to white light irradiation. The light intensities were set as 1500, 3500, or 5000 lx, with light exposure of 6, 12, or 24 h, after which periods oxidative damages to the RPE cells were assessed.

**Figure 6.** TER changes of the retinal pigment epithelium (RPE) cell monolayer during the cultivating processes.

#### *4.6. Fucoxanthin Pretreatment of RPE Cells*

In order to effectively improve the resistance of RPE cells to oxidative stress, the effect of fucoxanthin pretreatment time on the Nrf2-regulated antioxidant system was observed. Fucoxanthin or lutein was dissolved with a small amount of DMSO and added to the serum-free F12 medium at a final concentration of 20.0 μmol/L. Subsequently, the medium (200.0 μL) was added into the apical basolateral chamber. After incubation for 6, 12, or 24 h, the expressions of Nrf2 protein, as well as its regulated downstream antioxidant proteins, were analyzed.

#### *4.7. Protective Effect of Fucoxanthin against Visible Light-Induced Injury of RPE Cells*

After 24 h pretreatment with fucoxanthin, the medium of the apical chamber was replaced by a serum-free F12 medium containing 20.0 μmol/L fucoxanthin and 25.0 μmol/L DHA. Thereafter, the cell monolayer was subjected to light irradiation for 24 h at the light intensity of 3500 lx. The protective effects of FUCO on visible light-induced phagocytic disorder and oxidative damage in the RPE cells were subsequently observed. In order to verify whether fucoxanthin had enhanced the ability of RPE cells to resist photo-oxidative stress through the Nrf2 pathway, the cells were pretreated with the Nrf2 inhibitor ML385 (10.0 μmol/L) for 24 h, and the inhibitor was also added during the light exposure [21].

#### *4.8. Detection of ROS and MDA Content*

The intracellular ROS and MDA levels were measured as described in previous studies [23,42], with some modifications. The medium in the apical chamber was replaced by new medium containing 25.0 μmol of DCFH-DA. After 1 h incubation at 37 ◦C, the supernatant containing DCFH-DA was removed, and the cell monolayer was washed with Hanks' balanced salt solution (HBSS). The cell monolayer was then completely digested by RIPA lysis buffer, whereafter the cell lysate was added into a transparent black 96-well plate for fluorescence analysis. The fluorescence intensity was recorded using a microplate reader (Molecular Devices, San Jose, CA, USA) at 485 nm excitation and 530 nm emission. The MDA level in the cell lysate was measured according to the commercial kit's instruction, which was based on the thiobarbituric acid reactive substance (TBARS) assay.

#### *4.9. Detection of Intracellular SOD, HO-1, GCLC, GPx, NQO1, and TrxR Activity*

After the cell monolayer was washed with HBSS and fully lysed by RIPA lysis buffer, the expressions of antioxidative enzymes, including SOD, HO-1, GCLC, GPx, NQO1, and TrxR, in the cell lysate were determined, according to the manufacturer's protocols.

#### *4.10. Detection of Inflammatory Cytokines*

The inflammatory cytokines, including IL-6 and TNFα, were measured using commercial ELISA kits following the manufacturer's instructions.

#### *4.11. Measurement of Nucl-Nrf2*

The expression of Nucl-Nrf2 in the RPE cell monolayer was measured as described in a previous study, with some modifications [43]. Briefly, the differentiated RPE cell monolayer was treated with trypsin buffer at 37 ◦C for 10.0 min, whereafter the cells were collected and the nuclear proteins extracted using the nuclear extraction kit, according to the kit manufacturer's instructions. The protein concentration of each sample was normalized to total protein content in the cell pellet using the Bradford assay. Next, 20.0 μg nuclear protein was added into the 96-well plate based on the instructions of the Nrf2 transcription factor assay kit. The optical density was recorded using a microplate reader (Molecular Devices, San Jose, CA, USA) at 450 nm.

#### *4.12. Investigation of Phagocytosis of RPE Cells*

The phagocytosis of the RPE cells was determined according to our previous studies [1,23], with some modifications. Following light irradiation, the supernatant in the apical chamber was replaced by a serum-free medium containing fluorescent microspheres (1 × 107/mL). After 24 h incubation, the uningested microspheres were washed away with fresh serum-free medium, and, after being washed twice using HBSS, the cells were lysed by RIPA lysis buffer. Thereafter, the cell lysates were transferred to a black, clearbottomed 96-well plate, and the fluorescence intensity was recorded using a microplate reader (Molecular Devices, San Jose, CA, USA) at excitation/emission 360/420 nm. The phagocytic index was expressed as fluorescence intensity and presented as a percentage relative to the control.
