Figure 1

Phagocytosis of apoptotic neurons by microglia requires LRP/CRT signalling. (A) Example of flow cytometer phagocytosis assay data. Points represent individual cells with on the X axis staining for Alexa 488 IB4 (bound to BV2 cells only) and on the Y axis staining for TAMRA (bound to PC12 cells only). Co-staining (above the line) indicates BV2 phagocytosis of PC12s, which is increased when PC12s are apoptotic (right). (B) Fluorescent microscopy images of BV2 cells labelled with IB4 (green) showing BV2 that have phagocytosed TAMRA-labelled target cells (white hollow arrows) and unphagocytosed TAMRA-labelled target cells attached to BV2 cell surface (white solid arrow). DNA stained with Hoechst 33342 is shown in blue. (C) Cytochalasin D inhibits phagocytosis of apoptotic TAMRA-labelled PC12 by BV2 (flow data). (D) 10 μg/ml CRT-blocking antibody inhibits phagocytosis of apoptotic PC12 by BV2 (microscopy data). (E) Addition of 250 nM RAP to BV2 inhibits phagocytosis, but not when added to the apoptotic target PC12 cells (flow data). (C), (D) and (E) represent mean value from three separate experiments with two replicates per experiment. Error bars represent SEM. CRT, calreticulin; IB4, isolectin B4; LRP, low-density lipoprotein receptor-related protein; RAP, receptor-associated protein; TAMRA, 5-(and-6)-carboxytetramethylrhodamine succinimidyl ester.