Cd206 pe

7 days after CAP treatment, ASC were co-cultured with M0 macrophages. After 24, 48 or 72 h macrophages were harvested and labeled with CD45-APCVio770 (Milteny), CD206-PE (B&D) and HLA-DR-FITC (B&D) or their respective isotypes before flow cytometry analysis (LSR Fortessa, B&D). Mean fluorescent intensity (MFI) normalized isotype MFI were evaluated on living CD45 positive cells.

Each group of abdominal aorta blood samples containing macrophages were washed and treated with BD Pharm Lyse (BD Biosciences, San Jose, CA, USA). Inactive cells were excluded by trypan blue. The live cells were incubated with MHC-II-APC (BD Biosciences, San Jose, CA, USA) and CD-206-PE (BD Biosciences, San Jose, CA, USA). The fluorescent stained cells were analyzed by a BD LSR II flow cytometer using BD FACSDiva software (BD Biosciences, San Jose, CA, USA). And the fluorescent compensation and data analysis were performed by FlowJo 7.5 software (TreeStar, Ashland, OR). MHCII⁺CD206^- cells were indicated the M1 macrophages while MHCII⁺CD206⁺ cells were M2 macrophages. This assay was repeated at least three times.

To evaluate whether our treatments, nanocomplex (a drug-conjugated gold nanorods), the free drug (phosphatidylinositol 3-kinase (PI3Kα) inhibitor), and GNR: cholesterol-coated gold nanorods can do phenotype alteration by changing the expression of surface immune receptors or can modulate the intercellular communication; the surface marker expression was evaluated accordingly by flow cytometry.
First, 1 × 10⁶ cells/ml were harvested and collected by using 0.25%Trypsin EDTA (Gibco, USA). Then, cells were washed with PBS and centrifuged at 300 x g for 5 min. After that, cells were resuspended in stain buffer (BD, USA) and stained with the following flourscinated antibodies: CD11b-PE (ebioscience, USA), CD68-FITC (ebioscience, USA), CD14-PE-cy7 (BD, Biosciences, USA), CD206-PE (BD, Biosciences, USA), HLA-DR-PerCPCy5.5 (BD, Biosciences, USA), and CD45-FITC (BD, Biosciences, USA), in dark for 30 min at room temperature. Next, samples were washed with cell wash (BD. USA) and centrifuged at 300 x g for 5 min, followed by a resuspension step with 300 μl of PBS. Samples were acquired and analyzed with FACS DIVA software Version 8 on a FACS Canto II flowcytometer (BD, Biosciences, USA).

The tumor tissues were digested for one hour at 37 °C with collagenase and hyaluronidase (1 mg/mL) to prepare the single-cell suspension. The lymph nodes and spleen were added with PBS (1 mg/mL) and ground to prepare a single-cell suspension. The cells were incubated with PBS with 0.2% BSA on ice for 30 min, then washed with PBS. The cells were stained using CD8-PE, IFN-γ-PE-Cy7, CD11C-FITC, CD86-APC, CD206-PE, F4/80-FITC, CD44-FITC, or CD62L-APC (BD Biosciences, Franklin Lakes, USA) for flow cytometry assay according to a standard procedure.

Inflammatory cells in aortas were analyzed by flow cytometry as previously described^{25 (link)}. Single-cell suspensions were treated with Fc block, washed, and stained with CD45 percpCy5.5 (557235, BD), CD11b FITC (557396, BD), F4/80 BV421 (565411, BD), Ly6G APC (560599, BD), CD206 PE (141706, BD), CD3 BV421 (562600, BD), CD4 V500 (560782, BD), CD8 FITC (553030, BD), CD49b APC (558295, BD), NK1.1 PE (557391, BD), and their homologous isotype-matched negative controls (BD, Franklin Lakes, NJ). In the basis of a live gate, events were acquired on a Fortessa flow cytometer (BD) and analyzed. Detailed methods are described in the Data Supplement.