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. 2015 Aug 24;10(8):e0136708. doi: 10.1371/journal.pone.0136708

Fig 1. DIV crude supernatant induces IL-1β secretion independent of ADE.

Fig 1

(A) Flow-cytometric histogram overlay comparing CD14 expression levels in cells negative or positive for intracellular DENV E protein at 24 hpi. (B) Mobilized monocytes were inoculated with mock medium or DIV crude supernatant that had been incubated with 1 μg/ml mAb 5G22. At 1 hpi, cells were washed and resuspended in fresh medium. At 24 hpi, cells were washed and stained for surface expression (left panel) or intracellular expression (right panel) of DENV E protein with mAb 4G2 conjugated to AlexaFluor 647 and analyzed by flow cytometry. (C) Cumulative percentages of mobilized monocytes positive for intracellular DENV E protein at 24 hpi with DIV crude supernatant in the presence of increasing concentrations of mAb 5G22. (D) Measurement of secreted IL-1β by ELISA using 24-hpi supernatants from 1C. (E) Repeat of 1C using fresh, non-mobilized monocytes (one value per point). (F) Measurement of secreted IL-1β using supernatants from 1E (one value per point). (G) Measurement of DENV E-protein expression in mobilized monocytes at 24 hpi. Cells were inoculated with increasing doses of DIV crude supernatant with or without 1 μg/ml mAb 5G22. (H) Measurement of secreted IL-1β using 24-hpi supernatants from 1G. (I) Secreted IL-1β by mobilized monocytes at 24 hpi with mock supernatant or DIV crude supernatant derived from a second line of Vero cells. For all figures: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, and “n.s.” = not significant (p > 0.05). Test used: Two-Way ANOVA with Tukey’s post-test (I).