*4.3. Generation of Bone Marrow-Derived DCs (BMDCs)*

BMDCs were isolated and cultured using our improved method [61]. In brief, bone marrow cells were obtained from the tibias and femurs of C57BL/6 mice and cultured in complete medium (RPMI 1640 with 10% FBS, 1% streptomycin and penicillin (Invitrogen, Grand Island, NY, USA), 10 ng/mL GM-CSF and IL-4). The nonadherent cells were discarded and fresh medium was added after 60 h of culture. On day 6, nonadherent and loosely adherent cells were harvested and then cultured overnight. Only cultures with >90% cells expressing CD11c by FCM were used for the experiments.

#### *4.4. LPS-Induced Sepsis*

C57BL/6 mice, aged 6 weeks old, were randomly divided into five groups (*n* = 10/group), as described previously [31]. Astaxanthin (50, 100, and 200 mg/kg body weight) was orally administered for 2 days every 24 h; 48 h after the 1st oral administration, the mice were injected intraperitoneally with LPS (20 mg/kg body weight). Serum specimens were harvested 4 h after LPS treatment and were stored at −20 ◦C until use.
