Anti mouse cd16 32 monoclonal antibody

Immunized and challenged mice were killed under anesthesia conditions, to collect the nasal cavity by excision with scissors. The cells of the nasal mucosa were then collected by using sharp curettes in a dish filled with RPMI-1640 medium containing 2% NCS (Equitech Bio). These cell suspensions were filtered through 100 µm cell strainers (Corning). After treating with red blood cell lysis buffer for 1 min, cell samples were first incubated (15 min at room temperature) with anti-mouse CD16/32 monoclonal antibody (BioLegend; 93; 1:100) to avoid non-specific staining and with 7-aminoctinomycin D (7-AAD; BioLegend; 1:100) to detect dead cells and exclude them from the analysis. Then, the cell samples were stained with the following fluorescently labeled monoclonal antibodies for 30 min at 4 °C: fluorescein isothiocyanate – rat anti-mouse Ly6G (BioLegend; 1A8 1:100), APC rat anti-mouse CD45 (BD Biosciences A20; 1:100), APC-Cy7-rat anti-mouse CD11b (BioLegend M1/70; 1:100). Samples fluorescently labeled with monoclonal antibodies were fixed at 4 °C overnight with sterile PBS (Nacalai Tesque) containing 1% paraformaldehyde (Nacalai Tesque). Samples were examined by using a MACSQuant flow cytometer (Miltenyi Biotec, Auburn, CA, USA), and data analysis was performed with FlowJo 9.9 software (Tree Star, Ashland, OR, USA).

Periphery blood, spleens, and brains of mice treated with or without Pg were collected. Tissues of spleens and brains were ground and filtered through sterile cell filters. For blood and spleens, erythrocytes were lysed using RBC lysis buffer (CWBIO, Beijing, China) according to the instructions of the manufacturer and were then washed twice with PBS. A single-cell suspension of tissue was prepared. Anti-mouse CD16/32 monoclonal antibody (BioLegend) was used for blockage of Fc receptors. Dead cells were labeled with Zombie NIR Fixable Viability Kit (BioLegend). Cells were stimulated with Cell Activation Cocktail (BioLegend) and fixation/permeabilization was applied before intracellular staining. The antibodies were utilized for flow cytometry as follows: anti-mouse CD45 (clone 30-F11), anti-mouse CD11b (clone M1/70), anti-mouse CD3 (clone 145-2C11), anti-mouse CD4 (clone 145-2C11), anti-mouse CD8 (clone 53-6.7), and anti-mouse IFNγ (clone XMG1.2). All data were collected on a CytoFLEX (Beckman Coulter, USA) and analyzed with FlowJo software (version X, USA).