Calibur flow cytometry system

For cytometry, treated cells were blown down using cold phosphate-buffered saline (PBS, Invitrogen, San Diego, CA, USA), and 4 × 10⁵ cells were counted and suspended in 100 µL PBS supplemented with 4% FBS. Cells were then incubated with fluorescein isothiocyanate-anti-mouse CD86 (1:40; BioLegend, San Diego, CA, USA), allophycocyanin-anti-mouse CD206 (1:40; BD Pharmingen™, San Diego, CA, USA) at 4 °C for 30 min. After washing thrice with PBS, labeled cells were suspended in 100 µL PBS, and data were acquired via the fluorescence-activated cell sorting Calibur flow cytometry system (BD Bioscience, San Jose, CA, USA) and analyzed using the CytExpert software (Beckman Coulter, Inc., Brea, CA, USA).

Cell death was assessed by FITC Annexin V Apoptosis Detection Kit with 7-AAD (Cat. #:640,922) from BioLegend according to the manufacturer’s instructions and then analyzed with a flow cytometry calibur™ Flow Cytometry system (BD Biosciences). Data were analyzed and visualized using FlowJo software (flowjo.com).