2.4. Systemic and Pulmonary Immune Response
Five milliliters of venous blood were collected by using vacuum tubes containing EDTA K2 as anticoagulant. The whole blood analysis (white and red series) was performed using the automated system Sysmex 800i (Sysmex Europe GmbH, Germany). Immediately after whole blood analysis, the blood tubes were centrifuged at 1000g, for 7 minutes at 4°C. The serum was stored until the measurements of pro-inflammatory and pro-fibrotic mediators.
For analysis of pulmonary immune response, the levels of pro-inflammatory and pro-fibrotic mediators were measured in breath condensate, which has been collected using RT Tube (Respiratory Research, TX, USA) according to the manufacturer’s recommendations.
Thus, the levels of GM-CSF, TSLP, IL-4, IL-5 and TGF-beta was measured both in the serum as well as in the breath condensate by DuoSet ELISA kits (R&D Systems; MN, USA) through using a microplate reader Spectramax I3 (Molecular Devices, CA, USA).