Flow cytometry analysis for phagocytosis.

Flow cytometry analysis was employed to determine the phagocytosed level of fluorescent beads by macrophages (A–J). The time course of LT and PA treatments was 3 hours. In panels A, C, E, G, I, the X-axis represents particle/cell size (forward scatter) and the Y-axis represents the granularity of cells (side scatter). A and B: fluorescent beads only, C and D: macrophage cells only, E and F: beads + PA (85 ng/mL) treated cells, G and H: beads + LT (LF 150 pg/mL + PA 85 ng/mL) treated cells, I and J: beads + LT (LF 15 pg/mL + PA 85 ng/mL) treated cells. One representative diagram out of three experiments is shown (A–J). The R1 region in panels A, C, E, G and I reveals the population of the macrophage, which was specifically gated to obtain the graphs B, D, F, H, J, respectively. In panels B, D, F, H, J, the X-axis represents fluorescent intensity, which indicates the levels of macrophage-engaged beads, and the Y-axis represents the granularity of the cells (side scatter). The percentage of the cells with relatively high fluorescence intensity on the right panels of B, D, F, H, J, indicated the phagocytosed levels of beads by macrophages. In contrast, the percentage of cells on the left panels with relatively low fluorescence intensity indicates the number of cells without phagocytosed beads (B, D, F, H, J). The relative phagocytosis of macrophages was quantified by calculating the beads-phagocytosed (right panel: R)/non-phagocytosed (left panel: L) ratio of macrophages (K), the level of LF untreated (PA only) groups were normalized to 100%. The F, H, J labels in the columns of Fig. 3K indicate the respective conditions as showed in Fig. 3F, 3H and 3J, respectively (K). Error bars indicate standard deviations of three experiments. * P<0.05, ** P<0.01 compared to the PA (LF untreated) control groups. n = 6 (3 experiments with 2 replicates).