Quantifying Inflammatory Mediators in Mouse BAL

Mice were sacrificed by intraperitoneal (i.p.) administration of 90 mg/kg of body weight sodium pentobarbital. Bronchoalveolar lavage (BAL) was performed by washing the lungs with 3 mM EDTA in PBS (16 (link)) and cellular composition was determined by hemocytometer cell counts and differential counts of cytospins after staining with Quick-Diff solution (Siemens; Medical Solutions Diagnostics, Tarrytown, NY). Cell-free BAL fluid (BALF) was used to determine levels of IL-13 (4–500 pg/ml) and IFN-α (31.3–20.00 pg/ml) using ELISA kits (Ready-SET-Go; eBioscience, San Diego, CA), and levels of IL-1α (Minimal Detectable Concentration (MDC); 1.3 pg/mL), IFNγ (MDC; 0.8 pg/mL), TNFα (MDC; 1.9 pg/mL), IL-1β (MDC; 2.8 pg/mL), IL-10 (MDC; 2.1 pg/mL), IL-6 (MDC; 0.9 pg/mL), IFN-β (MDC; 4 pg/mL) using the LEGENDplex mouse inflammation panel (BioLegend, San Diego, CA). LEGENDplex panel was acquired on LSRII running FACS-Diva software (both obtained from BD Bioscience) and analyzed using BioLegend data analysis software. Results are from ≥4 mice per group (biological replicates) and 2 technical replicates per mouse.

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Shepardson K.M., Larson K., Johns L.L., Stanek K., Cho H., Wellham J., Henderson H, & Rynda-Apple A. (2018). IFNAR2 Is Required for Anti-influenza Immunity and Alters Susceptibility to Post-influenza Bacterial Superinfections. Frontiers in Immunology, 9, 2589.

Publication 2018

Quick-Diff solution Ready-SET-Go LEGENDplex mouse inflammation panel FACS-Diva software

Biological Body weight Bronchoalveolar lavage Bronchoalveolar lavage fluid Cell Diagnostics Edta Elisa Ifn α Ifnγ Il 10 Il 13 Il 1β Inflammation Lungs Mouse Sodium pentobarbital Tnfα

Corresponding Organization : Montana State University

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Variable analysis

independent variables

Intraperitoneal (i.p.) administration of 90 mg/kg of body weight sodium pentobarbital

dependent variables

Cellular composition of bronchoalveolar lavage (BAL) fluid, determined by hemocytometer cell counts and differential counts of cytospins
Levels of IL-13, IFN-α, IL-1α, IFNγ, TNFα, IL-1β, IL-10, IL-6, and IFN-β in cell-free BAL fluid (BALF), measured using ELISA kits and LEGENDplex mouse inflammation panel

control variables

Washing the lungs with 3 mM EDTA in PBS

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