Cytokine kits

China fine dust particulate matter (PM) (certified reference material No. 28) was purchased from the National Institute for Environmental Studies, Ibaraki, Japan. The organic solvents used in the experiments were of HPLC grade and were purchased from Sigma-Aldrich (St Louis, MO, USA). Silica gel 60 F254 TLC plates were purchased from Merck (Darmstadt, Germany). Silica (30–60 mesh), for open column preparation, was purchased from Sigma-Aldrich. Deuterated chloroform for NMR analysis was obtained from Cambridge Isotope Laboratories (Andover, MA, USA). The RAW 264.7 macrophage cell line was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM) with fetal bovine serum (FBS) and antibiotics (i.e., penicillin and streptomycin) purchased from GIBCO Inc. (Grand Island, NY, USA) were used as growth medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich. The cytokine kits used in the experiments were purchased from eBioscience (San Diego, CA, USA), R&D Systems (Minneapolis, MN, USA), BD Opteia (San Diego, CA, USA) and Invitrogen (Carlsbad, CA, USA). Santa Cruz Biotechnology (Santa Cruz, CA, USA) purchased antibodies were used for Western blotting.

IFN-γ and IL-4 secreted by primary splenocytes were detected using cytokine kits from Thermo Fisher Scientific, based on the manufacture’s protocol. The splenocytes were obtained from three immunized mice per group on day 35 (7 days after the third immunization). The mice were euthanized and the isolated spleens were sliced into small pieces and pressed through a 70 μm cell strainer (BD). The cells were washed with PBS and then the red cells were lysed. The splenocytes were resuspended in a T-cell medium (without supplement of IL-4) at 4 × 10⁶ cells/mL, and were transferred to 24-well plates (1.5 mL/well). The cells were incubated for 18 h at 37 °C in a 5% CO₂ atmosphere, with and without the CH401 peptide (20 μg/mL), and the supernatants were collected for the cytokine ELISA kits to quantify the levels of IFN-γ and IL-4.

DSS (MW 36,000–40,000 Da) was purchased from MP Biomedicals® (Santa Ana, CA, USA). The SOD Assay Kit was from Sigma–Aldrich® (St. Louis, USA). Cytokine kits were obtained from PeproTech® (PeproTech Brasil FUNPEC, Ribeirão Preto, SP, BR), protease inhibitor cocktail tablets were obtained from Roche® (Germany), and radioimmunoprecipitation assay (RIPA) buffer was obtained from Cell Signaling® (Danvers, MA, USA). All other chemicals and enzymes were purchased from Sigma–Aldrich® (St. Louis, MO, USA).

A superoxide dismutase (SOD) assay kit-WST® was purchased from Sigma-Aldrich. Cytokine kits were obtained from PeproTech® (PeproTech Brasil FUNPEC, Ribeirão Preto, SP, BR), protease inhibitor cocktail tablets were obtained from Roche® (Germany), and radioimmunoprecipitation assay (RIPA) buffer was obtained from Cell Signaling Technology®. All other chemicals and enzymes were purchased from Sigma-Aldrich® (St. Louis, USA).
The high-performance liquid chromatography system (HPLC) (LC-20 AT-Prominence, Shimadzu) coupled to a UV detector (Shimadzu, Serial no. L201550) was used. A biofreezer from the VIP Series by Sanyo was used. The spectrofluorometer was manufactured by Thermo Fisher Scientific® (Multiskan), who also supplied a Filizola® digital balance, with a capacity of 150 kg and 100 g accuracy and a stadiometer with a 2 m length and 0.1 cm precision.

Assay for Tumor necrosis factor a (TNF-a), Interleukin-1 (IL-1), Interleukin-10 (IL-10) was performed by ELISA using commercially available cytokine kits (Peprotech). Lungs, liver, kidney, spleen were homogenized in 1 ml lysis buffer containing 0.5% Triton X 100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl₂ and 1 mM MgCl₂ (pH 7.4). Homogenates were centrifuged at 400 g for 10 min and supernatants were used for estimation of cytokine levels. Appropriate antigen-affinity purified anti-mouse antibody pairs, detection reagents (TMB, BD biosciences) and mouse recombinant cytokines obtained from Peprotech were used as standards (capture antibodies: rabbit anti-mouse IL-1ß/IL-10/TNFa; detection antibodies: biotinylated rabbit anti-mouse IL-1ß/IL-10/TNFa). Absorbance was measured at 450 nm and results were expressed as pg/ml of cytokines released. All assays were performed in triplicates and performed thrice.