4.2.9. CLSM Observation of NF-Treated Cells

JAWS II cells (1.5 <sup>×</sup> 105) were cultured for 12 h in a 35 mm glass-bottom dish and subsequently washed with PBS and serum-free culture medium. Fluorescein-labeled peptide NFs were gently added to the cells, followed by incubation for 2 h at 37 ◦C with 5% CO2. After incubation, the cells were washed with PBS, and then incubated for 5 min with a solution containing LysoTracker Red DND-99 (50 nm) and Hoechst 33342, trihydrochloride, trihydrate (3.24 μM). LysoTracker Red DND-99 and Hoechst 33342 were used to stain the intracellular acidic compartments and nuclei, respectively. After staining, the cells were washed twice with PBS, then observed by CLSM using an FV10i microscope (Olympus, Tokyo, Japan).

4.2.10. Quantitative Expression Analysis of Co-Stimulatory Molecules and Cytokines from NF-Treated Cells

The expression of co-stimulatory molecules and cytokines was evaluated by specific immunostaining, as well as by ELISA. For immunostaining, JAWS II cells (2 <sup>×</sup> 105) were cultured for 12 h in a 24-well plate followed by washing with PBS containing 3% FBS and 0.05% NaN3, and then with serum-free culture medium. The DCs were pulsed with peptide NFs for 24 h, and then immunostained with a mouse monoclonal antibody for CD86 (a maturation marker), and subsequently analyzed by FCM to estimate their CD86 expression level. For quantitative analysis of TNF-α and IL-6 expression, the supernatants following the 24 h co-incubation of DCs with peptide NFs were collected and analyzed using an ELISA kit. The maturation of JAWS II cells cultured in medium with and without LPS (1 μg/mL) was evaluated as the positive and negative control, respectively. In addition, to compare the DC-activation ability between the NFs and heat-untreated building block peptides, JAWS II cells cultured with the peptides were also evaluated.
