Figure 3.

TmTNF-dependent endocytosis of anti-TNFs in dendritic cells. (A) Day 5 human CD14+ monocyte-derived DCs were treated with LPS for 2 h, incubated with pHrodo-conjugated anti-TNF and cellular uptake of labeled anti-TNF was monitored. Plasma membrane was stained using anti-MHC class I IgG (green), the nucleus (blue), and the internalized pHrodo-anti-TNF (red) were visualized using Amnis imaging flow cytometry. (B) Day 5 human DCs were treated with LPS for 2 h and cells were incubated either in the presence of pHrodo-conjugated matched isotype control IgG (red dotted histogram) or pHrodo-conjugated anti-TNF (blue filled histogram). Cellular uptake of the labeled antibodies was monitored for up to 4 h using flow cytometry. (C) To measure the kinetics of endocytosis of anti-TNF into acidic compartments in DCs, cells were either untreated (No LPS) or treated with LPS for 2 h. Cells were incubated with either human IgG1-pHrodo or anti-TNF-pHrodo antibodies. Endocytosis of the internalized anti-TNF was measured as an increase in fluorescence intensity by flow cytometry for up to 4 h.