Intracellular fixation and permeabilization kit

Samples were incubated with an Fc receptor blocker (CD16/32, BD Bioscience) to reduce nonspecific antibody binding. Freshly isolated samples were resuspended in staining buffer (R&D Systems) and stained with F4/80‐FITC (eBioscience) for 30 min at 4 °C. For intracellular staining, we used the Intracellular Fixation and Permeabilization Kit (BD Bioscience); this was used in accordance with the manufacturer’s instructions. Cells were then washed and stained with iNOS-APC (eBioscience) and CD206-PE (eBioscience). Flow cytometry was performed using a FACS Aria flow cytometer (BD Bioscience) and data were analyzed with FlowJo V10.6.2 software (TreeStar, Ashland, OR).

Single-cell suspensions were prepared from spleen or bone marrow samples for flow cytometry analysis. Red blood cells were lysed by ACK lysing buffer. For surface molecule detection, cells were stained with fluorochrome-conjugated antibodies for 10 min at room temperature in the dark. To detect intracellular molecules, intracellular fixation and permeabilization kit (BD Biosciences) or transcription factor staining buffer set (eBioscience) was used following manufacturer’s instruction. All FACS data were acquired on a LSRII instrument (BD Biosciences) and analyzed using Flowjo software (Tree Star).
To isolate different subsets of myeloid cells, cells were stained with CD11b-FITC and Ly6C-PE/Cy7 mAbs and subjected to cell sorting on a FACSAria (BD Biosciences). The purity of sorted cells was usually greater than 95%.