Bead mill homogenizer

Lungs were harvested from Nkx2.1creTpl2^flox/- mice, placed in 1 ml PBS and dissociated with a bead mill homogenizer (Qiagen). Lung homogenates were centrifuged at 500 x g for 5 min. Cytokines were quantified using a custom Procartaplex Immunoassay (Thermo-Scientific), IFN-α and IFN-β Luminescent ELISA kits (Invivogen) and Mouse Inflammation Cytometric Bead Array (CBA, BD Bioscience). IFN-λ3 (IL-28B) was measured using a colorimetric ELISA (Invitrogen). Hydroxyproline assay kit (Sigma, MAK357) was utilized to determine collagen levels in Nkx2.1creTpl2^flox/- lungs infected with influenza A/x31 for 8 days prior to harvesting.

Age-matched, six- to nine-week-old mice were sedated with 2.5% Avertin and intranasally infected with 50 μl of influenza A/x31 (10⁴ PFU) diluted in PBS. To determine lung viral titers, lungs were harvested on 1, 3, 5, and 7 days post infection (dpi), placed in 1 ml of PBS, and dissociated with a bead mill homogenizer (Qiagen). Virus titers were quantified by plaque assays (described below). To assess susceptibility to influenza infection, mice were infected with 10⁴ pfu of influenza A/x31 and observed over a 14-day period. Body weights were recorded daily, and mice exhibiting severe signs of disease or more than 30% weight loss were euthanized. For morbidity and mortality measurements, researchers were blinded to mouse genotype until the end of the experiment.

Age-matched, 6- to 8-week-old WT, Tpl2^-/-, Ifnar1^-/-, Ifnar1^-/-Tpl2^-/- or chimeric mice were anesthetized with 250 mg/kg Avertin (2,2,2-tribromoethanol) followed by intranasal infection with influenza A/HK-X31 (H3N2) in 50 μl PBS. Control mice were mock-infected with a similar dilution of allantoic fluid. To determine lung viral titers, whole lungs from WT and Tpl2^-/- mice infected with 10⁴ pfu of X31 virus were harvested on days 3, 5 and 7 pi. Lungs were placed in 1 ml PBS and dissociated with a bead mill homogenizer (Qiagen), and virus titers were enumerated by plaque assays. To assess susceptibility to influenza infection, WT and Tpl2^-/- mice infected with 10⁴ pfu of X31 virus were observed over a period of 14 days. Body weights were recorded daily, and mice exhibiting severe signs of disease or more than 30% weight loss were euthanized. To measure IFN and cytokine secretion, mice infected with 10⁶ or 10⁴ pfu of X31 virus were euthanized 3 or 7 days pi, and bronchoalveolar lavage fluid (BALF) or BAL cells were obtained by washing the lungs twice with 1 mL PBS. Cells were recovered by centrifugation of the lavage fluid for 10 min at 250xg. BALF from the first wash was used for quantitation of cytokine secretion. Cellular recruitment was assessed by quantifying total leukocyte recovery from both washes.