*4.10. Determination of the Antioxidant Enzyme Activity*

In vitro, after stimulation for 24 h with astaxanthin or astaxanthin plus LPS, DCs were collected and lysed with RIPA buffer. The GPx activity in the cell lysate supernatants or serum specimens was measured by the GPx assay kit (NJBC, Nanjing, China). In brief, 10 μL samples were mixed with 10 μL of GPx assay working solution and 176 μL of GPx assay buffer at 25 ◦C for 5 min. Then, 4 μL of cumene hydroperoxide initiated the reaction and absorbance was measured at 340 nm for 3 min.

The SOD activity in the cell lysate supernatants or serum specimens was measured by the SOD assay kit (NJBC, Nanjing, China) according to the manufacturer's instructions. The absorbance was measured at 450 nm.

The CAT activity was detected in samples by the CAT assay kit (NJBC, Nanjing, China). Briefly, the samples were treated with excess H2O2 for an exact time, and a substrate coupled with the remaining H2O2. After treatment with peroxidase, the absorbance was measured at 520 nm.

## *4.11. Western Blot*

Cells were washed once with ice-cold PBS and lysed with RIPA buffer. Protein concentrations were measured by the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts were separated on an SDS-PAGE and then transferred to the poly (vinylidene fluoride) (PVDF) membrane. After blocking with 5% dry powdered milk for 2 h, the membrane was immunolabeled with rabbit anti-mouse HO-1 or rabbit anti-mouse β-actin overnight at 4 ◦C, followed by goat anti-rabbit IgG-HRP for 1 h at room temperature. The membranes were developed in order to visualize the protein by adding an enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA). Autoradiograms were scanned and analyzed with Quantity One (Bio-Rad, Hercules, CA, USA) to quantify band densities.
