Fitc conjugated anti cd11b

The blood of mice was collected by removing eyeball into EDTA vacutainer tubes, and erythrocytes were lysed using red blood cell (RBC) lysis solution for 5 minutes at room temperature. Cells were centrifuged at 1000 g for 3 minutes to remove RBC lysis solution, and the leucocyte pellet was resuspended and washed with PBS. Leucocyte viability was counted via trypan blue exclusion method. Two‐colour flow cytometry analysis was performed on leucocytes. Unfractionated cells were stained with FITC‐conjugated anti‐CD11b (BD Biosciences) and Alexa Fluor^®647‐conjugated anti‐CCR2 (Biolegend, San Diego, CA). Cells were incubated at room temperature for 40 minutes, washed with PBS and stored at 4°C until the analysis. At least 50 000 cells were used per experiment.

Cell suspensions were prepared from mesenteric lymph nodes of KKAy mice according to previous report (Moro et al., 2015 (link)). Single-cell suspensions were stained with antibodies against the following cell surface antigens: fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (557396; BD, USA), phycoerythrin (PE)-conjugated anti-MHC-II (107608; Biolegend, USA), PE-CY7-conjugated anti-CD11c (558097; BD, USA), PE-CY5.5-conjugated anti-CD45.2 (109828; Biolegend, USA), and allophycocyanin (APC)-conjugated anti-F4/80 (123116; Biolegend, USA). Samples were detected on a NovoCyte flow cytometer (ACEA Biosciences, China), and the data were analyzed with NovoExpress flow cytometry analysis software (ACEA Biosciences, China).

Macrophages were trypsinized and Fc blocking (eBioscience 14–0161) was performed before staining with PE-Cy7-conjugatedanti-F4/80 (0.01 g/L, BioLegend 123114) or FITC-conjugated anti-CD11b (0.01 g/L, BD Pharmingen 553310). For negative control, cells were stained with the corresponding isotype control antibodies (BioLegend 400522,0.01 g/L and BD Pharmingen 553988, 0.01 g/L, respectively). Cells were analyzed using a LSRII Fortessa flow cytometer. Results were analyzed using BD Diva and FlowJo software.