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. 1998 Oct 5;188(7):1359–1368. doi: 10.1084/jem.188.7.1359

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Immature but not mature DCs efficiently phagocytose apoptotic cells. Freshly isolated blood monocytes were infected with live influenza A, PR/8 (Spafas Inc., Storrs, CT), labeled with the PKH26-GL fluorescent cell linker compound (Sigma Biosciences), and incubated at 37°C for 6–8 h, allowing apoptosis to occur. Macrophages, immature DCs, and mature DCs were dyed with PKH67-GL and added to the culture wells containing the apoptotic monocytes at a ratio of 1:1. Cells were analyzed by FACScan^® where double positive cells indicate uptake of the apoptotic cells by the various APCs (iii, vi, and ix). We used the various APCs alone to establish the proper settings (i, iv, and vii). Note that as the forward scatter of the APCs increased, the dying monocytes were excluded from the established region (ii, v, and viii). After 2 h, 80% of the macrophages, 50% of the immature DCs, and <10% of the mature DCs had engulfed the apoptotic monocytes (A). In an independent experiment, macrophages (squares), immature DCs (diamonds), and mature DCs (circles) were prepared, and cocultures with apoptotic monocytes were established as described above. FACS^® was performed at various time points. Percentage of phagocytosis was calculated based on the number of double positive cells (B).

Immature but not mature DCs efficiently phagocytose apoptotic cells. Freshly isolated blood monocytes were infected with live influenza A, PR/8 (Spafas Inc., Storrs, CT), labeled with the PKH26-GL fluorescent cell linker compound (Sigma Biosciences), and incubated at 37°C for 6–8 h, allowing apoptosis to occur. Macrophages, immature DCs, and mature DCs were dyed with PKH67-GL and added to the culture wells containing the apoptotic monocytes at a ratio of 1:1. Cells were analyzed by FACScan^® where double positive cells indicate uptake of the apoptotic cells by the various APCs (iii, vi, and ix). We used the various APCs alone to establish the proper settings (i, iv, and vii). Note that as the forward scatter of the APCs increased, the dying monocytes were excluded from the established region (ii, v, and viii). After 2 h, 80% of the macrophages, 50% of the immature DCs, and <10% of the mature DCs had engulfed the apoptotic monocytes (A). In an independent experiment, macrophages (squares), immature DCs (diamonds), and mature DCs (circles) were prepared, and cocultures with apoptotic monocytes were established as described above. FACS^® was performed at various time points. Percentage of phagocytosis was calculated based on the number of double positive cells (B).