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. Author manuscript; available in PMC: 2018 Feb 21.

Published in final edited form as: Virology. 2018 Jan 2;515:176–190. doi: 10.1016/j.virol.2017.12.025

Fig. 6 — The effects of Gas6-mediated efferocytosis on viral production from infected MT4CCR5 cells. (A) pHrodo Red-labeled MT4CCR5 and MT4CCR5/NL4-3 (2×10⁵ cells) 1, 2, 3, and 4 days post-infection were co-cultured with macrophages (2×10⁵) for 90 min in the absence and presence of Gas6 (1 μg/ml). The cells stained by APC-conjugated anti-CD14 antibody and pHrodo Red signals in the CD14-positive population were analyzed by flow cytometry. This experiment was repeated twice in triplicate, using cells from different donors for each experiment, and the results shown are averages and standard deviations of one representative triplicate experiment. (B) MT4CCR5/NL4-3 and MT4CCR5/Ada (1×10⁵ cells) were co-cultured with the same numbers of macrophages. The supernatants of the co-cultured cells were harvested 15 h later, and the titers of virus in the supernatants were titrated by GHOST (3) CXCR4+CCR5+ cells. The results shown are averages and standard deviations of two triplicate experiments, using macrophages from different donors. (C) MT4CCR5/NL4-3 and MT4CCR5/Ada (1×10⁵ cells) were co-cultured with the different cell numbers of macrophages. The supernatants of the co-cultured cells were harvested 15 h later, and the titers of virus in the supernatants were titrated by GHOST (3) CXCR4+CCR5+ cells. The results shown are averages and standard deviations of two triplicate experiments, using macrophages from donors K and L. (D) MT4CCR5/NL4-3 cells were labeled with CellTrace Violet and Ghost Red 780 2 days post-infection. The labeled cells (5×10⁵) were co-cultured with macrophages (5×10⁵) for 90 min in the presence of Gas6 (1 μg/ml). The cells were then stained with PE-conjugated anti-CD14 antibody and subjected to imaging flow cytometry. The representative images of live and dead MT4CCR5/NL4-3 cells internalized in macrophages are shown. (E) The results of Ghost Red 780 staining of the total population and the population internalized in macrophages of MT4CCR5/NL4-3 are shown as histograms. This experiment was repeated twice using cells from different donors.

Fig. 6 — The effects of Gas6-mediated efferocytosis on viral production from infected MT4CCR5 cells. (A) pHrodo Red-labeled MT4CCR5 and MT4CCR5/NL4-3 (2×10⁵ cells) 1, 2, 3, and 4 days post-infection were co-cultured with macrophages (2×10⁵) for 90 min in the absence and presence of Gas6 (1 μg/ml). The cells stained by APC-conjugated anti-CD14 antibody and pHrodo Red signals in the CD14-positive population were analyzed by flow cytometry. This experiment was repeated twice in triplicate, using cells from different donors for each experiment, and the results shown are averages and standard deviations of one representative triplicate experiment. (B) MT4CCR5/NL4-3 and MT4CCR5/Ada (1×10⁵ cells) were co-cultured with the same numbers of macrophages. The supernatants of the co-cultured cells were harvested 15 h later, and the titers of virus in the supernatants were titrated by GHOST (3) CXCR4+CCR5+ cells. The results shown are averages and standard deviations of two triplicate experiments, using macrophages from different donors. (C) MT4CCR5/NL4-3 and MT4CCR5/Ada (1×10⁵ cells) were co-cultured with the different cell numbers of macrophages. The supernatants of the co-cultured cells were harvested 15 h later, and the titers of virus in the supernatants were titrated by GHOST (3) CXCR4+CCR5+ cells. The results shown are averages and standard deviations of two triplicate experiments, using macrophages from donors K and L. (D) MT4CCR5/NL4-3 cells were labeled with CellTrace Violet and Ghost Red 780 2 days post-infection. The labeled cells (5×10⁵) were co-cultured with macrophages (5×10⁵) for 90 min in the presence of Gas6 (1 μg/ml). The cells were then stained with PE-conjugated anti-CD14 antibody and subjected to imaging flow cytometry. The representative images of live and dead MT4CCR5/NL4-3 cells internalized in macrophages are shown. (E) The results of Ghost Red 780 staining of the total population and the population internalized in macrophages of MT4CCR5/NL4-3 are shown as histograms. This experiment was repeated twice using cells from different donors.