Figure 1

Flow-cytometric analysis of the wild-type adult cochlea indicates the presence of innate and adaptive immune cells. Panels shows the gating strategy for flow-cytometric analysis of the immune cells in control spleen (A) and cochleae (B) of C57BL/6J mice. A gate was first set from the forward-scatter versus side-scatter dot-plot of all events in the area that corresponded to the size and granularity of lymphocytes using a spleen as a reference. The cells in the lymphocyte gate were then plotted as forward scatter versus Live-Dead stain. The cells in the live gate were then examined in a plot comparing the forward-scatter width and side-scatter area; cells inside the gate were determined to be single cells (singlets). The cells within the singlet gate were then plotted as a histogram of CD45 expression. Live CD45 + cells in the sample were further analyzed for B, T, NK, and myeloid cells and myeloid cells were further gated for macrophages and neutrophils depending on their surface-markers as described in methods section. Representative dot-plots from control C57BL/6 mouse spleen (C) and cochlea (D) shows various CD45 + immune cells including B, T, NK, and myeloid cells, macrophages and neutrophils with their respective surface expression of B220, CD3e, NK1.1, CD11b, CX3CR1, and Ly6G respectively. The numbers on the dot-plots shows the percentages of cells gated in the corresponding dot-plot. The numbers showed in the plots for B, T, NK, and myeloid cells are percentage of CD45 + cells while the numbers of macrophages and neutrophils are percentage of CD11b + cells, whereas the number for all immune cells in (E) are shown as the percentage of CD45 + cells. Panel F shows the absolute number of B cells (CD45 + B220 +), T cells (CD45 + CD3e +), NK cells (CD45 + NK1.1 +), myeloid cells (CD45 + CD11b +), macrophages (CD45 + CD11b + CX3CR1 +), and neutrophils (CD45 + CD11b + Ly6G + CD11c −) in the control cochleae.