Paracrine signaling enhances cytokine secretion by isolated cells.

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Paracrine signaling enhances cytokine secretion by isolated cells. Paracrine signaling enhances cytokine secretion by isolated cells. (A) Distribution of the single-cell secretion of TNF-α 4 hours after stimulation with LPS. Data are from 1329 cells combined from two independent experiments. The BT (black line) is 373 pg/ml. Cells that secreted TNF-α at a concentration above the BT are in black, whereas the top 5% of cells (in terms of amount of TNF-α that they secreted) are in red. Inset: Average secretion for all cells, cells above the BT (black), and the top 5% of cells (red). (B) Staining for intracellular TNF-α (red channel) in a cultured population of U937 cells that were incubated for 4 hours with LPS in the presence of brefeldin A. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole. Images are representative of two experiments. ICS, intracellular cytokine staining. (C) Schematic of the experimental setup for testing how the addition of a “uniform TNF-α” signal (1 ng/ml) compared to culturing the cells together for 4 hours (“paracrine mixing”) before they were isolated in the SCBC. (D) Measurement of the average amounts of the indicated cytokines that were secreted by single U937 cells cultured in the SCBC for 20 hours after they had been stimulated with LPS alone, LPS and TNF-α (1 ng/ml), or LPS followed by paracrine mixing. Data are means ± SEM of two independent SCBC experiments (n > 1280 cells for each condition). *P < 0.05 by t test. All cells above the DT were included in the analysis. The dotted line indicates the average amount of the indicated cytokines secreted in a cultured cell population (not shown for IL-6 and IL-10 because of differences in scale). Qiong Xue et al., Sci. Signal. 2015;8:ra59 Copyright © 2015, American Association for the Advancement of Science