Mouse inflammation kit

Of all mice, fresh feces were collected just before inoculation, snap-frozen in liquid nitrogen and stored at − 80 °C. Blood from the cardiac puncture was collected in heparin and immediately cooled. A lung and tongue were snap-frozen in liquid nitrogen and stored at − 80 °C. Furthermore, the other lung was homogenized in isotonic saline (4 ml per gram of tissue). Bacterial loads of the lung and blood were determined by serial dilution plated onto sheep-blood agar plates and incubated for 16 h at 37 °C, after which the CFU were counted. Following the plating, the blood was centrifuged at 3000 rpm for 10 min at 4 °C, in order to obtain the blood plasma. The lung homogenates were diluted 1:1 with a lysis buffer (1% (v/v) Triton X-100, 150 mM NaCl, 15 mM Tris, 1 mM MgCl(H₂O)₆, 1 mM CaCl₂(H₂O)₂, pH 7.4) including a protease inhibitor (complete protease inhibitor cocktail tablets, Roche, Basel, Switzerland) and incubated on ice for 30 min, followed by centrifugation at 4000 rpm, for 10 min at 4 °C, after which the supernatant was stored. In blood plasma and lung homogenate supernatant, interleukin (IL)-6, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1 and interferon (IFN)-γ levels were determined by a cytometric bead array multiplex (the Mouse Inflammation Kit, BD Biosciences, New Jersey, USA).

For cytokine analysis, Orbital blood was isolated from the mice for 400 μL, followed by centrifugation at 3500 rmp × 15 min, the supernatant were detected (Mouse Inflammation Kit, BD, US) in a BD FACSCalibur. Protein extracts from mouse tissues were determined by ELISA (Boster bio- Engineering Co., Wuhan).

Levels of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), macrophage chemotactic protein (MCP-1), interleukin-12 (IL-12), interferon-gamma (IFN-γ) and interleukin-10 (IL-10) were measured in lung and spleen homogenates. Mouse inflammation kit (BD Biosciences) was used for the simultaneous quantitation of these pro-inflammatory cytokines as previously described [13 (link)].

Twenty-three wild type, CypA-CMV, and CypA-SPC mice were inoculated i.n. with A/WSN/33(H1N1) virus at a dose of 3000 PFU. The mice were monitored daily for general behavior and clinical signs, including food intake, body weight, and inactivity. Five stable mice in each group were segregated for weighing. Three mice from each group were euthanized at 3, 5, 7, 9, 11, and 14 days post-infection (d.p.i.), and their lungs were collected, weighed, and homogenized using a QIAGEN TissueLyser II machine (30 cycles/s, 4 min) in 1 mL of cold PBS under sterile conditions. Solid debris was pelleted by centrifugation at 5,000 × g for 10 min, and the homogenates were used for virus titrations in MDCK cells as previously published51 (link). Their sera were used for cytokine detection by the cytometric bead array method using a Mouse Inflammation Kit (BD, USA). The lung index was calculated as the lung wet weight/body weight ×100. Additionally, a portion of each left lung lobe was fixed in 10% buffered formalin, embedded in paraffin, and 5-μm sections were stained with hematoxylin-eosin (H&E) and the monoclonal antibody against M1 protein for histopathological and immunohistochemical (IHC) analyses, respectively.