Anti mouse cd206 pe

FACS analysis was performed according to the previous reports (22 (link)). After perfusion of the kidneys with cold 1 × PBS, kidneys were removed, cut into fragments, and digested with 1 mg/ml collagenase and 0.1 mg/ml DNAase for 1 h at 37°C with intermittent agitation. Kidney fragments were passed through a 70-μm mesh (FALCON, 352350), producing single-cell suspensions. Approximately 1 × 10⁶ cells were stained for 30 min at room temperature with antibodies including anti-Mouse CD45-PE (8205729; Invitrogen), anti-CD11b-APC (8278517; BD Pharmingen), and anti-Mouse CD206-PE (4336334; BD Pharmingen), and resuspended in 1 × PBS. The suspensions were washed thrice with 1 × PBS, resuspended in 1 × PBS, and analyzed on High configuration Analysis Flow Cytometry (BECKMAN, CytoFLEX LX).

Macrophages were stimulated with IL4 (50 ng/ml, PeproTech, USA) for 24 or 48 h and then harvested and washed with cold 1 × PBS. Intracellular labeling with anti-mouse CD206-PE (BD Biosciences, USA) was performed using the Intracellular FIX&PERM Reagent set from MULTISCIENCES (China) according to the manufacturer's protocol. Cytofluorometric data were acquired with a BD FACS Canto II flow cytometer and analyzed using FlowJo 10.0.7.