M1270

Secreted Eotaxin levels were examined by ELISA as previously described using commercially available kits (for eotaxin/CCL11, R&D, catalog# MME00; for IL-4, R&D, catalog# M4000B; for IL-10, R&D, catalog#M1000B; for IL-12, R&D, catalog#M1270) according to vendor’s recommendations. For measuring eotaxin levels in the liver, cut a small slice of the tissue (~100 μg) and homogenize in ice-cold PBS with freshly added protease inhibitor cocktail (Sigma, P-8340). Centrifuge at 14000 g at 4 °C for 15 min. Remove and aliquot the supernatant for ELISA. Data were normalized by cell number (for supernatant collected from primary cell culture) or tissue weight (for liver homogenates).

Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the concentrations of IL-1β (#MLB00C, R&D Systems), IL-18 (#7625, R&D Systems), IL-1α (#MLA00, R&D Systems), tumor necrosis factor (TNF; #MTA00B, R&D Systems), IL-6 (#M6000B, R&D Systems), and IL-12 p70 (#M1270, R&D Systems) in cell culture medium or serum according to the manufacturer's instructions.

Concentrations of mouse IL-23 (R&D Systems, #DY1887) and IL-12 (R&D Systems #M1270) were determined in spinal cord supernatants 7 and 14 days post-immunisation by ELISA, as per the manufacturer’s guidelines.

De-immortalized DCs were seeded at 1*10⁵ cells/flask in 25 cm² cell culture flasks and allowed to settle overnight in a humidified atmosphere at 37 °C and 5% CO₂. Media were then removed and fresh media (5 ml) was given with either no additions (controls), 33% Cu-doped TiO₂ NPs at 40 µg/ml (NP condition) or addition of maturation factors IL1β (25 ng/ml) TNFα (50 ng/ml) and IFNγ (1000 units/ml) (all from PeproTech; this is the “classical” activation scheme) for 24 h. Media were then removed and cells were incubated with soluble recombinant CD40L (BioTechne) at 16 µg/ml for 24 h after which the supernatants was collected and used to determine IL12p70 levels by ELISA (M1270, R&D Systems). Cells were centrifuged at 2500 rpm for 6 min after which cells were stained with anti-Ccr7 antibody (PE-anti-CD197 (Ccr7), clone 4B12, Thermo Fisher Scientific) for 30 min at 4 °C. The cells were then centrifuged again, and resuspended in PBS after which they were run on the ImageStream X Mark II (Merck, Belgium) for analysis.

De-immortalized DCs were seeded at 1*10⁵ cells/flask in 25 cm² cell culture flasks and allowed to settle overnight in a humidified atmosphere at 37 °C and 5% CO₂. Media were then removed and fresh media (5 ml) was given with either no additions (controls), NP conditions or classical activation scheme for 24 h. For migration, DCs (25*10³ in 25 µL volume were seeded on the membrane surface of 5 µm pore ChemoTx 96-well plate (NeuroProbe, Gaithersburg, MD) and incubated for 90 min at at 37 °C, with 10 ng/ml 6C-kine present in the bottom chamber, after which the number of migrated DCs in the bottom chambers were counted. To determine the IL-12p70-producing ability of migrated DCs, recombinant soluble CD40L was added directly to the bottom chambers, containing the migrated DCs, for 24 h, after which the IL12p70 was determined from the supernatant by ELISA (M1270, R&D Systems).

Human or mouse IL-12-ABP protein (0.25 mg/mL) was mixed with 2.5 mg/mL Alhydrogel in TBS and incubated for 30 minutes to form ANK-101 or mANK-101 complexes. Complexes were incubated at 37°C in TBS with or without 40% serum and 1 mM phosphate. At various times, samples were centrifuged to pellet the Alhydrogel and free IL-12-ABP was measured in the supernatant using an ELISA against mouse IL-12 (R&D Systems, m1270) or human IL-12 (R&D Systems, 24910 for capture; BioLegend, 508801 for detection).