Luminex xmap system

Eight days pi, mice were killed and tracheotomy was performed. The mouse lungs were flushed with 1 ml of PBS, and the retained BAL was centrifuged at 400 g for 5 min at 4 °C. The recovered supernatants were collected and stored at −80 °C until assessed for cytokine concentration, and the cell pellet were resuspended in 200 μl of 10% buffered formalin. Total cell numbers were counted using a hemocytometer. Cytokines in BAL supernatants were quantified with the Luminex xMAP system using a MILLIPLEX MAP mouse cytokine immunoassay (MCYTOMAG-70K; Millipore, Billerica, CA, USA) according to the manufacturer protocol. In brief, beads coupled with anti-CCL2, anti-IL-6, anti-interferon-γ, anti-RANTES, anti-IL-15 and anti-TNF monoclonal antibodies were sonicated, mixed and diluted 1:50 in assay buffer. For the assay, 25 μl of beads were mixed with 25 μl of PBS, 25 μl of assay buffer, and 25 μl of BAL supernatant, and incubated overnight at 4 °C. After washing, beads were incubated with biotinylated detection antibodies for 1 h and the reaction mixture was then incubated with streptavidin-phycoerythrin (PE) conjugate for 30 min at room temperature, washed and resuspended in PBS. The assay was analyzed on a Luminex 200 instrument (Luminex Corporation, Austin, TX, USA) using the Luminex xPONENT 3.1 software.

Serum levels of cytokines including IL‐1β, IL‐5, IL‐6, IL‐7, IL‐12p40, IL‐12p70, G‐CSF, IFN‐γ, and TNF‐α were detected using the Luminex^® xMAP™ System (Millipore Corporation, Germany). The assays were conducted in strict accordance with the operating instructions of the instrument. CRP and PCT were measured during the first 48 hours after onset of fever. Serum CRP was measured through nephelometric method (Siemens BNII, Cardiophase, Germany) and PCT was measured by Cobas800 (Roche, Switzerland) based on electrochemical luminescence technology.