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).