Apc conjugated anti cd206

Flow cytometry was used to assess microglial markers. A Neural Tissue Dissociation Kit (MACS) was used to homogenize the brains of the mice according to the instructions (Miltenyi Biotec). A Percoll gradient was used to collect monocyte‐enriched cells.38 Cells were labeled with APC‐Cy7‐conjugated rat antimouse CD45 (BD, 557659), FITC‐conjugated rat anti‐CD11b (BD, 553310), PerCP‐Cyanine5.5‐conjugated F4/80 (Invitrogen, 45‐4801‐82), APC‐conjugated anti‐CD206 (Invitrogen, 17‐2061‐82) and BV510‐conjugated anti‐CD86 (BD, 563077) and suitable isotype controls according to the instructions (eBioscience). Before staining CD206, the cells were treated with Fixation/Permeabilization Concentrate (eBioscience, 00‐5123‐43, 00‐5223‐56) for 30 minutes and then incubated with APC‐conjugated anti‐CD206 in permeabilization buffer (eBioscience, 00‐8333‐56) for 30 minutes and analyzed on a FACSVerse cell sorter and studied with FlowJo software.

PE-conjugated anti-F4/80 (BD Pharmingen, CA, USA, 565410) was used as a pan-macrophage cell surface marker. To further distinguish macrophage M1 and M2 polarization states, PE-Cy™7-conjugated anti-CD86 (BD Pharmingen, CA, USA, 560582) was chosen to mark the M1 phenotype, and APC-conjugated anti-CD206 (Invitrogen, CA, USA, 17-2061-82) was chosen to mark the M2 phenotype. M1 phenotype was labelled as F4/80⁺CD86⁺, whereas M2 phenotype was labelled as F4/80⁺CD206⁺ (30 (link), 36 (link)).
RAW264.7 cells or macrophages isolated from the spleen of mice were suspended in cold stain buffer (FBS); this was followed by incubation with PE-conjugated anti-F4/80 and PE-Cy™7-conjugated anti-CD86 or APC-conjugated anti-CD206 antibodies on ice in a dark room for 30 min. Then we washed these cells three times with FBS and examined using a CytoFlex flow cytometer (Beckman Coulter, CA, USA) within 2 h. Flow cytometric images were analyzed using CytExpert software. Gating strategy is shown in the Supplementary Figure S2.