Specific elisa

Cells plated in duplicates in 6 well plates were cultured for 6, 12, and 24 hours. At the indicated time point, the culture supernatant was collected and concentrated using Amicon ultra-centrifugal filters (3KDa cutoff). The protein retenate was reconstituted up to 0.5ml with PBS. TGF-β1 levels in the supernatant were assessed using a specific ELISA (R&D systems, Minneapolis, MN), according to the manufacturer's recommended protocol. The total protein concentration was determined by BCA. The TGF-β1 levels were normalized to the total protein content of each sample. Results were expressed as TGF-β1 pg/ml concentration.

For protein analysis in lung tissue, lung biopsy tissue from IPF/UIP patients or from the normal margins of lung tumor resections, or lungs from mice, were homogenized in antiprotease buffer (Roche Diagnostics) and processed as previously described (46 (link)). BAL was taken from the mice by flushing the lungs with sterile PBS, while serum was prepared from a postmortem blood draw. Whole lung lobes were dissected for histological and biochemical analysis. Cytokine levels were measured by bead based LuminexÔ analysis or specific ELISA (R&D Systems). In some instances, mediator levels were normalized to protein content, which was determined using a standard Bradford protein assay. For gene analysis, total RNA was obtained using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Gene expression levels were quantitated using real time RT-PCR (Applied Biosystems), according to the manufacturer’s protocols. Undetectable expression was set as zero for comparisons. Transcript levels of genes of interest were normalized to β-actin or 18S mRNA.

In all experiments proliferation was assessed by CFSE staining (5μM). After gating on CD4⁺ and/or CD8⁺ T cells, the percentage of proliferating cells in each population was determined. For cytokine analysis, supernatants were taken at the end of the time cultures and IFN-γ production or IL-10 was assessed by specific ELISA (R&D Systems). Levels of other cytokines were detected using a cytometric bead array (eBioscience). For intracellular cytokine staining, cells were washed and stimulated with PMA (50 ng/mL) and ionomycin (2000 ng/mL) for 5 hours. BFA (5μg/mL) was added for the last 3 hours. Afterwards, the staining of cell surface markers was performed. Cells were washed, fixed, permeabilized (intracellular cytokine staining kit, eBioscience), and stained for detection of IFN-γ.

Mice were pretreated with 100 μl of PBS or rhAAT variants (50 μg per mouse i.p., n = 20 per experiment) for 3 h, then treated with 1 mg/kg LPS (i.p.). Blood samples (20 μl) were collected from the tail vein at 1.5, 3, and 24 h later, and serum was separated by centrifuge; sera were analyzed for TNFα concentrations using specific ELISA (R&D Systems).

The inflammatory cell populations in the effluents were analyzed by flow cytometry using a FACS Canto II cytometer (BD Biosciences). The following monoclonal antibodies (BioLegend) were used: anti-CD11b (clone M1/70), anti-F4/80 (clone BM8), anti-CD19 (clone 6D5), anti-Gr1 (clone RB6-8C5), and anti-TCRb (clone H57-597). Data were analyzed using FlowJo software (Tree Star). Inflammatory cytokines IL-6, IL-10, IFNγ, tumor necrosis factor(TNF)-α, and MCP-1 were analyzed using a bead-based flow cytometry assay (CBA kit, BD Biosciences, San Jose, CA, USA), whereas TGF-β and VEGF-A were analyzed using specific ELISA (R&D Systems, Minneapolis, Minnesota) according to the manufacturer’s instructions.

Blood from the cubital vein was taken under fasting conditions immediately before PCIs into serum seperator tubes. Samples were allowed to clot for 30 minutes (min), were then centrifugated at 1500g for 15min and stored at −80°C until use. G-CSF was measured by specific ELISA (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Laboratory determinations were performed by investigators that were blinded to clinical characteristics and patients' outcome.