Macrophage Phenotypic Profiling by FACS

Macrophages were stimulated (section 2.3 above), washed and blocked with Fc blocking Ab (BD Biosciences) in fluorescent-activated cell sorting (FACS) buffer (PBS, 0.1% NaN₃, 1.0% FBS for 15 min at 4°C [11 (link), 12 (link)]. The cells were washed and stained with fluorochrome-conjugated antibodies (Abs) (SOCS1-Alexa Fluor® 488, SOCS3-Alexa Fluor® 647, NOS2-PE and MRC1/CD206-Alexa Fluor® 680 (Santa Cruz Biotechnology, Dallas, TX, USA)) for 30 min at 4°C, and then washed, fixed with 2% paraformaldehyde solution (PFA) for 20 min at 4°C. Data were acquired on a BD FACS Canto II flow cytometer (BD Bioscience) with at least 1 × 10⁵ events for each sample and analyzed using FCS Express 6 FLOW (De Novo Software, Pasadena, CA, USA).