Allophycocyanin streptavidin

RSV was conjugated with Dynabeads Intact Virus Enrichment (#10700D, Thermo Fisher Scientific) in accordance with the manufacturer’s instructions in such a way that the RSV was 1.0 × 10⁷ PFU per 1 µL of beads. The RSV-Beads were then incubated with 70 µL of nasal wash from immunized mice for 0.5 h at room temperature, followed by staining with FITC-conjugated anti-mouse IgG (1:100; #115-096-062, Jackson ImmunoResearch) or biotin-conjugated anti-mouse IgA (1:1000; #1040-08, SouthernBiotech) and allophycocyanin–streptavidin (1:1000; # 20-4317-U100, Tonbo Biosciences, San Diego, CA, USA). Antibody binding was assessed by measuring the intensity of fluorescent labeling by flow cytometry, and the mean fluorescent intensity of each group was measured and expressed with the value for RSV-Beads alone set to 1. Flow cytometric analysis was performed by using an Attune NxT flow cytometer, and all data were analyzed using the FlowJo software package (Supplemental Fig. 3).

NTHi strain 76 was grown on a chocolate agar plate overnight at 37°C, and cells were collected in PBS and centrifuged at 10,000g for 5 min at 4°C, after which the cell pellet was diluted into FACS buffer. These NTHi cells (2 × 10⁸ cfu) were then incubated with nasal wash (equivalent to 50 μg protein input) for 1 h at room temperature, followed by staining with biotin-conjugated anti-mouse IgA (BioLegend, San Diego, CA, USA) and allophycocyanin–streptavidin (Tonbo Biosciences, San Diego, CA, USA). Flow cytometric analysis was performed by using an Attune NxT flow cytometer (Thermo Fisher Scientific).