Anti mouse cd16 32 pe

Flow cytometry was used to detect the expression of CD16/32 and CD206 in microglia cells as previously described [31 (link)]. In brief, 100 μl microglia cell suspension (1 × 10⁶ cells) was aliquoted into 1 ml Eppendorf tubes. The anti-mouse CD16/32 PE (0.125 μg, Biolegend, CA, USA) and anti-mouse CD206 (MMr) FITC (0.125 μg, Biolegend, CA, USA) were added into the EP tubers after 0.5 h of incubation at 4 °C, The microglia cells were washed with PBS twice and resuspended in 300 μl 1 × PBS solution. Analyses were performed on Becton-Dickinson FACS Calibur (Becton Dickinson, Bedford, MA). Mean fluorescence intensity was measured.

For analysis of FcγR expression in macrophages, 1 × 10⁶ cells from the spleen of ITP mice were stained with anti-mouse CD11b-FITC (Biolegend), anti-mouse CD16/32-PE (Biolegend), and anti-mouse CD64 (Biolegend) monoclonal antibodies for 30 min at 37°C in the dark. Purified human CD14+ cells were exposed (continuous treatment) to DMSO or ML604440 (300 nM) or ONX-0914 (30 nM) for 24 h. Cultured human monocytes/macrophages (1 × 10⁶) were incubated (RT, 30 min in the dark) with a cocktail of anti-human CD14-APC (Biolegend), anti-human CD16-FITC (Invitrogen), and anti-human CD64-PE (Biolegend) antibodies. Data analysis was carried out using a FACS Calibur flow cytometer equipped with Kaluza Flow Cytometry Analysis Software (Beckman Coulter).