Ready set go kit

IFNγ, TNFα and IL-2 quantifications were performed on cell culture supernatants with Ready-SET-Go! kits (eBioscience) according to manufacturer’s instructions. MIF was quantified by ELISA (Biomatik) using 48 h cell culture supernatants from naïve and anti-CD3 antibody-activated CD4+ T lymphocytes. Two hundred thousand lymphocytes were seeded in MultiScreen-IP 96-wells (Merck Millipore), pre-coated with mouse IFNγ from ELISPOT Ready-SET-Go (eBiosciences) in the presence or absence of LPS for 24 h. ELISPOT membranes were revealed following the manufacturer’s instructions and data was acquired and analyzed with the ImmunoSpot analyzer (CTL Europe GmbH).

Blood was collected for separation of serum at various time points after various treatments as specified for experiments. Serum IL-6, TNFα and IL-10 levels were estimated using a protocol recommended by the manufacturer of Ready-Set-Go kits (eBioscience). Actual values for cytokines were extrapolated based on known standards. Samples with absorbance values below detection limit in each assay were labelled as ND.

The levels of TNF‐α, IL‐6, IL‐8, and MCP‐1 in the conditioned media were determined by enzyme‐linked immunosorbent assay using Ready‐SET‐Go kits (eBioscience, San Diego, CA). For measurement of TNF‐α and IL‐8 production by U937 macrophages, samples were diluted at a ratio of 1 : 10 and 1 : 100, respectively; for measurements of IL‐6 and MCP‐1 production by hPdLFs samples were used undiluted and for measurement of IL‐8 production samples were diluted at a the ratio of 1 : 5. The detection limit for all cytokines was 2 pg ml⁻¹.

Mouse bone marrow-derived dendritic cells (BM-DC) derived from naïve BALB/c mice (8 weeks of age; n = 3) were prepared as previously described [34 (link), 39 (link)]. Briefly, bone marrow precursors isolated from femurs and tibias were seeded at 2 x 10⁵ cells/ml in RPMI 1640 culture medium containing 10% FCS, 150 μg/ml gentamycin, and 20 ng/ml mouse GM-CSF (Sigma-Aldrich, Germany) and incubated for 8 days. BM-DC (10⁶ cells/well) were stimulated with formalin-inactivated B. longum strains Bl 7952 and Bl 372 (10⁷ CFU/well), Pam3CSK4 (1 μg/ml), ultrapure LPS (1 μg/ml, InvivoGen, USA) or left untreated for 18 h. The levels of IL-10, IL-12p70, IL-6 and TNF-α were analysed in supernatants of stimulated cells by ELISA using Ready-Set-Go! kits (eBioscience, USA) according to manufacturer’s instructions. For cell surface marker analysis, BM-DC were labelled for 30 min at 4°C with anti-mouse FITC-conjugated CD11c, APC-conjugated MHC II and PE-conjugated CD40, CD80 or CD86 monoclonal antibodies (eBioscience, USA). The data were acquired on a BD FACSAria III flow cytometer (BD Biosciences, USA) and analysed with FlowJo software 7.6.2 (TreeStar, USA).

Due to slow viral clearance in aged mice, lung lysates were obtained at different time points, day 6–8 post infection from the lung extracts in 1.5 ml of RPMI media. Virus titers were determined by calculating 50% egg infectious dose (EID₅₀) after incubation at 37ºC for 3 days in the 10-day-old embryonated chicken eggs. Interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) ELISA was performed using Ready-Set-Go kits (eBioscience, San Diego, CA).

Tumor necrosis factor (TNF-α), IFN-γ, IFN-α, and IL-6 levels were detected by human enzyme-linked immunosorbent assay (ELISA) Ready-Set-Go Kits (eBioscience). Concentration of the cytokine was expressed as amounts per mL of plasma or supernatant.
The levels of MMPs (Bio-Plex Pro human MMP Panel) were detected by the Luminex enzyme immunoassay (Luminex, TX) and analyzed in vitro.