Elisa immunoassay kit

At the end of the timed ventilation protocol and after completion of hemodynamic measurements, mice were exsanguinated via sectioning of the left carotid artery. Arterial blood gases were determined in five WT_(LPS+MV) group mice. Bronchoalveolar lavage (BAL) was performed with four separate 0.5 mL aliquots of saline at 20 °C followed by lung fixation (paraformaldehyde 4%, inflation pressure 25 cmH₂O for 1 min) and paraffin embedding. The total cell count of BAL fluid was determined from a fresh BAL fluid specimen using a Malassez hemocytometer. The BAL fluid was centrifuged (1600 rpm, 7 min at 4°C), the cell pellet was diluted in saline, and differential cell counts were done in four WT_(LPS+MV) mice and four IL-6KO_(LPS+MV) mice on cytocentrifuge preparations (Cytospin 3; Shandon Scientific, Cheshire, UK) stained with Diff-Quick (Baxter Diagnostics, McGaw Park, IL). Cell-free supernatants were stored at −80°C for subsequent duplicate measurements of total protein (Bio-Rad Protein Assay) and cytokine concentrations [macrophage inflammatory protein 2 (MIP-2), tumor necrosis factor α (TNF-α) and IL-6 using an ELISA immunoassay kit, (R&D systems, Abingdon, UK) performed in accordance with manufacturer’s instructions.