Figure 5.

Flow cytometry determined that primary human monocytes can survive and differentiate into macrophages at physiological temperature of the zebrafish in vitro. (a) Schematic showing that human primary monocytes were cultured at physiological temperature of zebrafish (28.5 °C) or at physiological temperature of humans (37 °C) with or without differentiation by cytokine human macrophage colony stimulating factor (H-M-CSF) for 5, 8, and 11 days. Bottom panel micrographs show the morphology of cells in the presence or absence of cytokine at each temperature. (b) Graphs (mean ± SD) showing fraction of migrated cells in response or absence of isolated zebrafish astrocytes in a transwell assay. *P = 0.0410 by Tukey’s multiple comparisons post-test, n = 3 membranes/condition for astrocytes isolated on a single day. (c) Primary monocytes from 5 donors were either cultured at physiological temperature of humans (37 °C) or at physiological temperature of zebrafish (28.5 °C) with or without H-M-CSF. Plot (mean ± SD) of average survival calculated for primary human monocytes/macrophages that survived over the course of 5 days were normalized to the original seeding density. Significantly more cells survived at 28.5 °C vs. 37 °C in the presence or absence of H-M-CSF by two-way ANOVA. (d) Human primary monocytes were cultured at physiological temperature of zebrafish (28.5 °C, left panels) or at physiological temperature of humans (37 °C, right panels) with cytokine human macrophage colony stimulating factor (H-M-CSF) for five, eight and 11 days. Expression of CD14 was assessed by flow cytometry. Light grey curve indicates control where shift in dark grey curve indicates expression. (e) Cells positive for CD14 expression were then assessed for markers, CD86, CD163 and CD206 by flow cytometry at each temperature and time point.