Figure 3. tdTomato positive inner ear cells from Gfi1^Cre mice consist of CD326 + CD45- cells and CD326-CD45+ cells.

(A) Cochlear single cell suspensions from four day old Gfi1^Cre/+;ROSA26^CAG-tdTomato mice were analyzed for the expression of CD326 and CD45 in the tdTomato positive cell population. One representative FACS analysis is shown. From left to right: (1) Forward and side scatter of the dissociated cells. The analysis was focused on the marked population to exclude cellular debris; (2) Gating for doublet discrimination; (3) CD326 vs tdTomato expression. The tdTomato cells (3.3% of total singlet cells) consist both of CD326 positive and negative cells; (4) Gating on the tdTomato positive cells (marked in a box in 3), the cells are divided to two distinct populations: CD326 + CD45− which represent HCs, and CD326-CD45+ which represent immune cells. (B) The mean + SD percentage of each population was summarized from 5 individual mice (43.4 ± 14.7% CD326 + CD45− HCs, 49.6 ± 13.2% CD326−CD45+ immune cells). (C) Single cell suspensions from spleen and cochleae of wild-type 4 day old C57BL/6 mice were analyzed for immune cell surface markers by FACS. 5 populations were identified within CD45+ gated cells in the cochlea: P1: 3.1% CD11b+Gr1+ granulocytes; P2: 81.3% CD11b+Gr1− monocytes/macrophages; P3: 0.8% CD3+T cells; P4: 3.4% CD3-DX5+NK cells; P5: 0.4% B220+ B cells (D). Data is representative of one out of two experiments using pooled cochlear cells from 5 and 7 mice, respectively. SSC-A = side scatter-A, FSC-A = forward scatter-A, FSC-H = forward scatter-H.