*4.2. Reagents*

Astaxanthin (mol wt 596.84), LPS derived from *Escherichia coli* 026: B6, FITC-Dextran (mol wt 40,000) and Cobalt protoporphyrin (CoPP, a HO-1 inducer) were from Sigma-Aldrich. Alexa Fluor 647-Dextran (mol wt 10,000) was from Thermo Fisher. Carboxyfluorescein succinimidylester (CFSE) and RPMI 1640 medium were from Invitrogen. Fetal bovine serum (FBS) was from Hyclone. Recombinant CCL19, GM-CSF, and IL-4 were from Peprotech. CCK-8 kit was from Beyotime. CD4+ T cell isolation kit was from Miltenyi Biotech. Fluorescent-labeled anti-mouse mAbs, PerCP-Cy5.5 CD69, FITC-MHCII, PE-CD40, PE-CD80, FITC-CD86, PE-CCR7 or respective isotype controls, were from BD PharMingen. Alexa Fluor 647 HO-1 or respective isotype was from Abcam. PE-Nrf2 or respective isotype was from Cell Signaling Technology. Tin protoporphyrin IX (SnPP, a HO-1 inhibitor) was from MedChemExpress.

#### *4.3. Generation of DCs*

Male C57BL/6 mice, 4–6 weeks old, were from the Animal Research Center of Yangzhou University (Jiangsu, China). The mice were housed under specific pathogen-free conditions for at least 1 week before use. DCs were isolated and cultured as our improved method [53]. Briefly, bone marrow cells were extracted from the tibias and femurs of mice, and then cultured in complete medium (RPMI 1640 supplemented with 10% FBS, 1% streptomycin and penicillin, 10 ng/mL GM-CSF and 10 ng/mL IL-4). After 60 h of culture, medium was gently discarded and fresh medium was added. On day 6, non-adherent and loosely adherent DC aggregates were harvested and sub-cultured overnight. On day 7, only cultures with >90% cells expressing CD11c by flow cytometry (FCM) were used.

#### *4.4. Cell Viability Assay*

The cytotoxicity assay of astaxanthin with different doses was performed in DCs using the CCK-8 kit in accordance with the manufacturer's instructions. Briefly, 5 × 103 cells were cultured in 96-well plate. After treatment, 10 μL CCK-8 was added to each well, and the cells were incubated for an additional 1 h. The absorbance was measured at 450 nm, and the results were compared as a percentage of the control group.

#### *4.5. Cytokine Assay*

In vitro, the DCs were incubated with astaxanthin and/or LPS for 24 h. Next, the levels of TNF-α, IL-6, and IL-10 in the culture supernatants were measured by using ELISA kits (eBioscience) and were performed according to the manufacturer's instruction.
