Phagocytic Activity of Differentiated Cells

Differentiated LGCs, FBGCs and osteoclasts were detached using Accutase (StemCell Technologies, Cat # 07920) and were filtered through a 70 μm nylon mesh (VWR, Cat # 732‐2758). For DNA staining, cells were labeled with 10 μg/ml Hoechst 33342 (Sigma‐Aldrich, Cat # B2261) in PBS supplemented with 1% FBS and 2 mM EDTA. Cells were then centrifuged and re‐suspended in cold PBS. Phrodo red S. aureus bioparticles (Invitrogen, Cat # A10010) were added according to the manufacturer's instructions and incubated for 30 min at 37°C. Cells were re‐centrifuged, fixed in 0.4% formaldehyde and analyzed with ImageStream (Amnis Corporation). Fluorescence was measured by ImageStream at 375 nm (Hoechst) and 581 nm (Phrodo red S. aureus bioparticles). Based on their size and Hoechst staining, cells were categorized as mononuclear and multinucleated cells. The mean fluorescence of Phrodo red S. aureus bioparticles was measured as a readout of phagocytic activity of the cells. Results were analyzed with Ideas v5 Software (Amnis Corporation).