Rat anti mouse cd11b apc cy7

PE-CF594 rat anti-mouse CD24 (562477, 2.5:100), BB515 rat anti-mouse I-A/I-E (MHCII 565254, 1.25:100) and PE rat anti-mouse IgG1 κ isotype control (554685, 5:100) were purchased from the BD Bioscience company. PerCP-Cy5.5 rat anti-mouse CD45 (45-0451-82, 1:100), APC rat anti-mouse IgG2b κ isotype control (17-4031-82, 1.25:100) and APC rat anti-mouse CD206 (17-2061-82, 1.25:100) were purchased from eBioscience. The PE rat anti-mouse α7AChR (sc-58607PE, 5:100) was purchased from Santa Cruz. APC-Cy7 rat anti-mouse CD11b (101226, 2.5:100), PE-Cy7 hamster anti-mouse CD11c (117318, 1.25:100) , Brilliant Violet 421 rat anti-mouse CD86 (105032, 1:100) , Brilliant Violet 421 rat IgG2a κ isotype control (400536, 1:100) , APC rat anti-mouse CD24 (138506, 3.75:100) and Brilliant Violet 421 mouse anti-mouse CD64 (139309, 1:100) were purchased from the Biolegend. Lipid droplet dye, BODIPY™ 493/503 (D-3922, 1:200) was purchased from the Invitrogen. Rabbit anti-mouse CD68 antibody (BA3638, 1:100) was purchased from Boster. Rabbit anti-mouse F4/80 antibody (GB11027, 1:800) was purchased from Servicebio.

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).