Cd163 apc

To identify each macrophage phenotype, THP-1 cells were seeded at a density of 8 × 10⁵ cells/well in a 6-well plate and polarized into M0, M1, and M2 macrophages using the aforementioned methods. The cells were washed with phosphate-buffered saline (PBS) and harvested using trypsin-EDTA. The cells were washed using wash buffer (BD Biosciences, San Jose, CA, USA) and stained with antibodies. The following antibodies were purchased from BD Bioscience: human CD86-PE-Cy7 and CD163-APC.
To test peptide affinity, THP-1 cells were seeded at a density of 5 × 10⁶ cells/well in 6-well plates. The cells were polarized as described above. After incubation, cells were washed with PBS and harvested using trypsin-EDTA. The cells were further washed with a wash buffer (BD Biosciences) and stained with 100 nM FITC-conjugated melittin or melittin fragments (Genscript) for 1 h at 4 °C. The cells were washed three times with the wash buffer. All data were analyzed using the FACSLyric system (BD Biosciences) and FlowJo software (Tree star, San Carlos, CA, USA).

For the differentiation and polarization of isolated human monocytes into M1 and M2 macrophages, cells were cultured for 7 days at 37 °C in a 5% CO₂ atmosphere in complete medium composed of RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution in the presence or absence of different cytokines. Monocytes were seeded in 96- or 6-well culture plates at 2 × 10⁵ cells/well or 1 × 10⁶ cells/well, respectively, and the following cytokines were added for 7 days: 50 ng/mL GM-CSF (for M1 polarization) or 50 ng/mL M-CSF (for M2 polarization). At day 6, 100 ng/mL LPS was added to M1 polarized macrophages and 20 ng/mL IL-4 was added to M2 polarized macrophages. Seven days later, cells were detached from plates and stained with a cocktail of conjugated antibodies as follows: 10 µL CD14-FITC, 5 µL CD16-APC-H7, 5 µL CD80-PE-Cy5, 5 µL CD163-APC, and 5 µL CD200R-PE from BD Biosciences. Data acquisition was performed on 10,000 cells with constant PMT values on an FACS Canto II cytometer (BD Biosciences). Data analysis was carried out using Diva software (BD FACSDiva v9.0software, BD).