Affinipure goat anti mouse igg igm h l

For fixation, 4% paraformaldehyde was applied to the cell slides at room temperature for 15 min, followed by three washes with phosphate-buffered saline (PBS). Cells were permeabilized with PBS containing 0.3% Triton X-100 (Sigma-Aldrich Co. LLC, Saint Louis, MO, USA) twice for 15 min at 4 °C. The cells were then incubated with 2% bovine serum albumin (Bionovas biotechnology Co., Ltd., Toronto, Ontario, Canada) in PBS for 30 min. Slides were then incubated at 37 °C for 1 h with anti-cytokeratin 19 antibodies (Abcam plc., Cambridge, UK) diluted 1:100 with blocking solution (final concentration 10 μg/mL), followed by three washes with PBS. Fluorescein isothiocyanate-conjugated AffiniPure goat anti-mouse IgG + IgM (H+L; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), diluted 1:100 to a final concentration of 15 μg/mL, was applied as the secondary antibody and incubated at 37 °C for 30 min, followed by three washes with PBS. For nuclear staining, slides were incubated with 4′, 6-diamidino-2-phenylindole (Life Technologies, Grand Island, NY, USA) for 15 min at room temperature, followed by three washes with PBS. Finally, the slide was dipped into PBS containing Evans blue for background counterstaining to show the localization of the cells.

Lymphocytes were obtained from the pneumonic lung by bronchoalveolar lavage (BAL) and adherent cells were removed by incubating on plastic for 1 h at 37 °C. Single-cell preparations of spleen were enriched for CD8 T cells by panning on tissue culture plates coated previously with AffiniPure goat anti-mouse IgG + IgM (H + L) (Jackson ImmunoResearch Labs). CD8⁺ T-cell responses to each candidate epitope were tested via intracellular cytokine staining. Enriched lymphocytes from spleens or bronchoalveolar lavage of mice infected 10 days previously with IAV were incubated with 10 U/ml IL-2 (Roche) and 1 μg/ml Golgi-plug (BD Biosciences) in the presence or absence of 1 μM synthetic peptide in 96-well plates and cultured for 5 h at 37 °C and 5% CO₂. Cells were then stained for surface expression of CD8α and intracellular IFN-γ and TNF. Data was acquired by flow cytometry (FACSCanto, BD Biosciences) and analysis was performed using Flowjo version 9.6 (FlowJo LLC, Ashland). Single viable live CD8⁺ lymphocytes were gated for analysis (Supplementary Fig. 7). A response was deemed positive if the test response (with background subtracted) was equal to or greater than 2× the average background values.

The clinical specimens of throat swab, nasopharyngeal swab, sputum, bronchoalveolar lavage (BAL) or pleural effusion were inoculated into human embryonic lung cells (HEL), rhabdomyosarcoma cells (RD) and Madin-Darby canine kidney cells (MDCK) while those of blood, cerebrospinal fluid (CSF), urine, stools or rectal swabs were inoculated into adenocarcinoma human alveolar basal epithelial cells (A549), monkey kidney cells (MK-2), RD cells and HEL cells. Cultures were maintained in minimal essential media containing antibiotics, incubated at 33 C, and rotated at 12 revolutions per hour. The inoculated cell cultures were maintained and observed for the presence of cytopathic effects (CPE) for at least 4 weeks. Respiratory viruses, including human adenovirus (HAdV), human metapneumovirus, human parainfluenza virus types 1, 2, and 3, influenza A and B viruses, Cytomegalovirus, HSV-1, HSV-2 and respiratory syncytial virus in CPE-positive cases were further identified by indirect immunofluorescence assay (IFA) with D3 Ultra DFA respiratory virus screening and identification kit (Diagnostic Hybrids, Inc., Athens, OH, USA). Anti-HSV 1 monoclonal antibody (ARGENE) react with cells infected with herpes simplex type 1 and display clear staining with Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Mouse IgG + IgM (H + L) (Jackson).