Bio plex pro mouse cytokine group 1 panel 23 plex

After lung mechanics analyses, BAL fluid was collected via the intratracheal instillation and recovery of 1 mL of PBS. Total cells in the recovered BAL fluid were counted using a hemocytometer and the number of neutrophils, eosinophils, macrophages and lymphocytes were quantified from modified Wright-Giemsa-stained cytospin preparations of BAL cells (200 cells/slide). Lung lobes were separated and processed as follows: flash frozen in liquid nitrogen for cytokine and chemokine analyses by Lincoplex according to manufacturer’s instructions (Bio-Plex Pro™ Mouse Cytokine Group I Panel 23-Plex, BioRad) or gene expression analyses using quantitative RT-PCR, or inflation-fixed with 10% buffered formalin and paraffin embedded for histology32 (link)33 (link). For Lincoplex analyses, the following cytokines and chemokines were measured (the limit of detection): IL-1α (2 pg/mL), IL-1β (7 pg/mL), IL-2 (3 pg/mL), IL-3 (2 pg/mL), IL-4 (3 pg/mL), IL-5 (2 pg/mL), IL-6 (2 pg/mL), IL-9 (15 pg/mL), IL-10 (2 pg/mL), IL-12p40 (2 pg/mL), IL-12p70 (4 pg/mL), IL-13 (9 pg/mL), IL-17A (1 pg/mL), EOTAXIN (148 pg/mL), G-CSF (1 pg/mL), GM-CSF (7 pg/mL), IFNγ 6 pg/mL), CXCL1 (3 pg/mL), MCP-1 (14 pg/mL), MIP-1α (24 pg/mL), MIP-1β (2 pg/mL), CCL5 (5 pg/mL), TNFα (6 pg/mL).

Serum was stored at −80°C until use. ELISA kit detecting mouse IgG was obtained from Thermo Fisher Scientific, mouse anti-dsDNA from Alpha Diagnostic International. For the kit detecting mouse anti-dsDNA, serum was diluted 1:100. Assays were performed according to the manufacturer's instructions. Cytokine levels were measured using Bio-Plex Pro™ Mouse Cytokine Group I Panel 23-plex (BioRad) according to the manufacturer's instructions.

Lungs were collected from the aged mice that survived viral challenge and pushed through 40 µm cell strainers (VWR, Radnor, PA) in 1 ml of RPMI 1640 media (Mediatech, Manassas, VA). Lung lysates were clarified by centrifugation at 10,000 x g for 10 min and cytokines were measured using Bio-Plex Pro mouse cytokine group 1 23-plex panel (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. Cytokine concentrations were normalized per total protein measured using bicinchoninic acid (BCA) assay with bovine serum albumin as the standard (Thermo, Massachusetts, USA).