Rab0477

NP effect on the release of five inflammatory mediators in LPS+ RAW264.7 was further quantified using mouse TNF-α, IL-1β and IL-10 ELISA kits purchased from Sigma-Aldrich (RAB0477, RAB0274 and RAB0245); and mouse IL-6 and PGE₂ ELISA kits purchased from abcam (ab222503 and ab133021). In brief, cells were seeded into six well culture plates at a density of 3 × 10⁵ cells/well and cultured for 24 h, then, cells were activated with LPS in RAW264.7 culture medium (1 µg mL⁻¹ for IL-1β quantification and 500 ng mL⁻¹ for TNF-α, IL-6, PGE₂ and IL-10) and exposed simultaneously to NPs (CLX-10, TNX-5 and DEX-15 at 0.50 mg mL⁻¹). Following a 24 h incubation, supernatants were collected and stored at −20 °C until use. Levels of TNF-α, IL-1β, IL-6, PGE₂ and IL-10 in cell culture supernatants were determined by the corresponding ELISA kit according to the protocol recommended by the manufacturer. LPS-stimulated and unstimulated cells treated with PBS were used as controls (LPS+ and LPS-, respectively). Experiments were performed using five replicates per formulation and results were expressed as mean ± SD. ANOVA were performed comparing tested NPs and LPS+ at significance levels of p < 0.05, p < 0.005 and p < 0.001 and comparing NPs with each other at significance levels of p < 0.05, p < 0.005 and p < 0.001.

Serum samples and cell culture supernatant were subjected to ELISA assays to measure pro-inflammatory cytokines. Following the ELISA kit manufacturer’s instructions, the contents of TNF-α (RAB0477, Sigma-Aldrich), IL-6 (RAB0308, Sigma-Aldrich) and IL-1β (RAB0274, Sigma-Aldrich) were determined.

Blood samples were collected from the left ventricle on the 8th day after DSS treatment. A protein-based enzyme-linked immunosorbent assay (Elisa) was used to detect the levels of cytokines (RAB0274; RAB0477; RAB0308; RAB0245, Merck, USA). In brief, after the antibody was coincubated overnight with the plate, the sample to be tested was added, followed by a reaction solution for coloration, and the level of cytokine was detected by colorimetry. Limulus test kit (EC644405; Xiamen Houshiji, Ltd., Xiamen, China) was used to detect LPS.

Levels of TNF-α, IL-6 and other factors in the BALF were analysed by ELISA using commercially available kits (RAB0477, RAB0308, Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Levels of TNF-α, IL-2 and other factors in murine serum were analysed by ELISA using commercially available kits (#560,484, Cell Signaling Technology Company, MA, USA) according to the manufacturer’s instructions.