Figure 1. uPAR is required for the internalization of apoptotic cells in vitro.

(A) Absence of uPAR expression in uPAR-deficient macrophages. Macrophages were isolated from WT and uPAR^−/− mice (n=3) and pooled together, respectively, their expression of uPAR was measured by Western blot analysis (upper panel) and flow cytometry (lower panel). The data are representative of three experiments.(B) Internalization of apoptotic cells. Peritoneal macrophages from WT and uPAR^−/− mice were incubated with apoptotic cells at 37°C for 15 minutes, followed by treatment with or without 0.04% trypan blue (TB) to quench the fluorescence of unengulfed apoptotic cells. Phagocytosis of apoptotic cells was analyzed by flow cytometry. Association is indicated in percentage as binding and ingestion (TB:-) and ingestion (TB:+). **, p<0.01(i). Quench of fluorescence signal of CFDA-SE-stained apoptotic cells by TB was confirmed by flow cytometry (ii). (C) Viable or apoptotic neutrophils (5×10⁵) from WT or uPAR^−/− mice were incubated with adherent WT or uPAR^−/− macrophages (5×10⁴) on 96-well plate at 37°C for 15 minutes. After macrophages were treated with TB, phagocytosis was analyzed by flow cytometry. *, p<0.05; ***, p<0.001.