Bio plex pro mouse group 1 23 plex panel

Levels of cytokines and chemokines in BAL fluid were determined with the Bio-Plex Pro Mouse Group I, 23-plex panel (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instructions^{49 (link)}. Groups included PBS (n = 2), RSV (n = 11), PG-RSV (n = 10), and PG-CAT – RSV (n = 12) for day 1, and PBS (n = 7), RSV (n = 8), PG-RSV (n = 11), and PG-CAT – RSV (n = 11) for day 2. The panel included the following cytokines with the lower limit of quantitation (LLQ): IL-1α (1.84 pg/ml), IL-1β (10.36 pg/ml), IL-2 (3.72 pg/ml), IL-3 (1.55 pg/ml), IL-4 (6.98 pg/ml), IL-5 (3.57 pg/ml), IL-6 (0.74 pg/ml), IL-9 (6.89 pg/ml), IL-10 (2.95 pg/ml), IL-12 p40 (1.53 pg/ml), IL-12 p70 (1.62 pg/ml), IL-13 (47.2 pg/ml), IL-17 (2.65 pg/ml), granulocyte-macrophage colony-stimulating factor (GM-CSF) (21.2 pg/ml), gamma interferon (IFN-γ) (1.84 pg/ml), tumor necrosis factor alpha (TNF-α) (5.8 pg/ml), G-CSF (5.1 pg/ml), Eotaxin (257.9 pg/ml), KC (3.2 pg/ml), MCP-1 (22.4 pg/ml), macrophage inflammatory protein 1α (MIP-1α) (256.2 pg/ml), MIP-1β (3.33 pg/ml) and RANTES (2.78 pg/ml). Data is presented as a mean of three experiments (PBS n = 7, and infected groups n ≥ 8).

Cytokines, chemokines, interferons, and elastase were all measured using BALF collected at day one p.i. Total proteins were measured using BALF collected at days one and five p.i. Levels of cytokines and chemokines in the BALF were determined with a Bio-Plex Pro Mouse Group I 23-plex panel (BioRad Laboratories, Hercules, CA, USA). Interferon (IFN)-α and IFN-β were measured by ELISA, following the manufacturer’s protocol (PBL Biomedical Laboratories, Piscataway, NJ, USA). Total protein concentrations were determined using the Bradford method (BioRad Laboratories, Hercules, CA, USA). Neutrophil elastase was measured using a neutrophil elastase ELISA kit (R&D Systems, Minneapolis, MN, USA). Absorbance for all microplate assays was measured on a SpectraMax 190 microplate reader (MDS Analytical Technologies, Sunnydale, CA, USA).

Levels of cytokines and chemokines in BALF were determined at day one p.i. with a Bio-Plex Pro Mouse Group I 23-plex panel (Bio-Rad Laboratories, Hercules, CA, USA). CXCL2 was measured using a Mouse MIP-2 (CXCL2) ELISA kit (ThermoScientific, Fredrick, MD, USA). Neutrophil elastase was measured using a neutrophil elastase ELISA kit (R&D Systems, Minneapolis, MN, USA). HMBG1 and lactate dehydrogenase (LDH) were measured in BALF as markers of cell damage. Under normal conditions, HMGB1 is found within the nucleus where it stabilizes nucleosome formation and facilitates transcription factor binding. During necrosis of the cell, HMGB1 can be acetylated allowing for its secretion into the extracellular environment [30 (link)]. LDH is a stable cytoplasmic enzyme that is rapidly released upon damage to the plasma membrane [31 (link)]. HMGB1 was measured using a HMGB1 ELISA kit (IBL International GMBH, Hamburg, Germany). LDH activity was measured using the Colorimetric Lactate Dehydrogenase (LDH) Activity Assay kit (Abcam, Cambridge, UK). All kits were processed according to the manufactorers instructions. Absorbance for all microplate assays was measured on a SpectraMax 190 microplate reader (MDS Analytical Technologies, Sunnydale, CA, USA).