This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTrackerTM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than necroptosis.
[Background] Studying the phagocytic uptake of necroptotic cells by BMDCs in vitro, is a preliminary step in the examination of immunogenic cell death models (Obeid et al., 2007). Efficient uptake will allow the phagocyte to cross-present antigens to leukocytes and thereby create an immune reaction towards the dead target cells. In this protocol we make use of a CellTrackerTM dye. This type of dye may be toxic in certain concentrations, which may vary depending on which cell type is used. Thus, we recommend users to first find the optimal concentration of the dye for the target cell in use. Optimally, the CellTrackerTM dye itself should not induce any cell death, but should label the target cells so that they become easily separable from the CD11c-positive BMDCs.
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