Anti mouse cd11c percp cyanine5

The adipose tissues and bronchus of each group of lungs were removed. The lungs were then cut into pieces and digested in RPMI 1640 medium (VivaCell, Shanghai, China) containing 10% FBS (Sperikon Life Science & Biotechnology Co., Ltd., Chengdu, China) and collagenase VIII (250 U/mL; Sigma Aldrich, Saint Louis, MO, USA) at 37 °C for 30 min. Cell surface staining was performed by incubating the cells with the indicated antibodies on ice for 15 min. Cells were washed and analyzed on an LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) using FlowJo software (BD Biosciences, San Diego, CA, USA).
To measure the percentages of CD4⁺ and CD8⁺ cells among CD3⁺ mononuclear cells, the cells were incubated with anti-mouse CD3e-FITC (1:1200, eBioscience); anti-mouse CD4-APC (1:400, eBioscience); anti-mouse CD8a-PE (1:1600, BioLegend); anti-mouse CD45R-PerCP-Cyanine5.5 (1:800, eBioscience) for 20 min at room temperature.
To measure the percentages of macrophages and dendritic cells, cells were incubated with anti-mouse I-A/I-E-PE/Cyanine7 (1:3200, BioLegend); anti-mouse F4/80-FITC (1:100, eBioscience); anti-mouse CD11b-eFluor™ 450 (1:800, eBioscience); anti-mouse CD11c-PerCP-Cyanine5.5 (1:200, eBioscience); anti-mouse Ly-6C-APC (1:800, eBioscience) for 20 min at room temperature.

All cell suspension samples were adjusted at 10⁶ cells per 50 μl before running the flow cytometry. Cells were then incubated for 10 min at 4°C with anti-mouse CD16/32 (Biolegend, USA) for blocking Fc receptors. Surface molecules of DCs were stained for 30 min at 4°C with the following antibodies: anti-mouse CD11c Percp-cyanine 5.5 (eBioscience, USA), anti-mouse lineage brilliant violet 421, anti-mouse B220-APC-Cy7, anti-mouse CD40-APC, anti-mouse CD80-PE, anti-mouse CD86-FITC, and anti-mouse MHC-Ⅱ PE-cyanine 7 (Biolegend, USA). Treg cells were stained with surface markers: anti-mouse CD4-FITC (Biolegend, USA) and anti-mouse CD25-APC (eBioscience, USA). The intranuclear Foxp3 of Treg cells was stained with anti-mouse Foxp3-PE (eBioscience, USA) following cell fixation and permeabilization with the transcription factor buffer set (BioLegend, USA). Cellular fluorescence was assessed with FACS Canto II (BD Biosciences, USA) and data were analyzed with FlowJo software (version 10.2, TreeStar, USA) [29 (link)].