Elisas

IL-10 in cell culture supernatants was measured using ELISAs according to the manufacturer’s instructions (Biolegend, San Diego, CA, USA). Detection limits for IL-10 were 2000 pg/mL.

ELISAs (Biolegend, San Diego, CA) were performed on the homogenized cranial and middle lobes of the right lung to analyze concentrations of specific proteins, including TNF-α; monocyte chemoattractant protein-1 (MCP-1), a chemoattractant responsible for monocyte and macrophage recruitment; CXCL-1, seen in inflammation or wound healing; and IL-1β and IL-6, both mediators of inflammatory responses. The cranial and middle lobes from control- and PM-exposed animals and standards from the R&D Systems ELISA kits (1000 μg/mL to 7.8 μg/mL) were prepared and examined in duplicate in 96-well plates using a SpectroMax plate reader (Molecular Devices, Sunnyvale, CA). Duplicate readings were averaged. All concentrations were normalized to total lung protein and reported in pg of specific protein per mg of lung tissue.

Twenty-four and forty-eight hours after cell plating, SNs were collected from Treg and ASC monocultures, as well as direct and indirect cocultures. SNs were frozen and stored at −80 °C until analysis. Concentrations of 48 mediators: sCD40L, EGF, eotaxin, FGF-2, FLT-3L, fractalkine, G-CSF, GM-CSF, GROα, IFNα2, IFNγ, IL-1α, IL-1β, IL-1RA (IL-1 receptor antagonist), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-1/CCL2, MCP-3, M-CSF, MDC/CCL22, MIG/CXCL9 (monokine induced by IFNγ), MIP-1α/CCL3, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES/CCL5, TGFα, TNFα, TNFβ and VEGF-A were measured with Human Cytokine/Chemokine/Growth Factor Panel A 48 Plex Kit (Merck) and analysed with Luminex (MAGPIX, Merck Millipore) according to the manufacturer instructions. In addition levels of, IL21 and IL35 were measured with ELISAs (BioLegend and Elabscience, respectively).

Following sacrifice, whole blood was collected by cardiac puncture and allowed to sit overnight at 4°C. Serum was separated by centrifugation at 10,000 × g for 2 min, aliquoted, and stored at −80°C until analysis. Concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were determined by single-plex enzyme-linked immunosorbent assays (ELISAs; BioLegend, San Diego, CA).

TNF-α, IL-6 and IL-12 levels in cell supernatants were evaluated by commercially available ELISAs (Biolegend, California) according to the manufacturer's instructions.

Cytokine release assays were performed by co-culture of 10⁵ T cells with 10⁵ target cells per well in 96-well round bottom plates in a final volume of 200 ul of RPMI complete media. After ∼24 hrs, supernatants were assayed for presence of IFN-γ and IL-2 using ELISAs, according to manufacturer's instructions (Biolegend). IFN-γ, IL-2, IL-4, IL-10, MIP-1A and TNF-α cytokines were measured by flow cytometry with Cytometric Bead Array (BD Biosciences), based on protocols from the manufacturer. All cytokine data are represented as a mean of triplicate wells +/− SEM.