Figure 2
Analysis of dissociated retinal cells and sorted cells by flow cytometry. Retinal cells were analysed using fluorescence-activated cell sorting. The x-axis shows fluorescent of PerCP-Cy5 to detect 7-Amino-Actinomycin D (7-AAD) fluorescence which labels dead cells and the y-axis shows fluorescence of AmCyan-A to detect cyan fluorescent protein (CFP) fluorescence. (a) Analysis of dissociated retinal cells of Thy1-CFP mice which manifest CFP in retinal ganglion cells. Cells which possess relatively strong CFP fluorescent without 7-AAD fluorescence, whose CFP fluorescence was considered not to be autofluorescence, were contained in the area 1 (0.04–0.12% of total cells). (b) Analysis of dissociated retinal cells of wild-type mice. No cells were contained in the area 1 (CFP-positive). (c) Re-analysis of the sorted cells by gate RGCs (see Method and Fig. S1). 88.9–93.4% of the sorted cells were included in area 1 (CFP-positive). (d) The sorted cells were stained with anti-brain-specific homeobox/POU domain protein 3A (Brn3a, green) and anti- paired box genes 6 (Pax6, red) antibodies. Nuclei were counter stained with 4’,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 50 µm. (e) mRNA expression of synaptosomal-associated protein 25 (Snap25), tubulin, beta 3 class III (Tubb3), RNA binding protein with multiple splicing (Rbpms) and rhodopsin (Rho) in cells sorted by gate RGCs or gate PR (see Methods and Supplementary, Fig. S1b) were visualised with the Integrated Genome Browser. Snap25, Tubb3 and Rbpms were highly expressed in Gate RGCs and not in Gate PR while Rho was highly expressed in Gate PR and not in Gate RGCs (black arrows). RGC: retinal ganglion cell, PR: photoreceptors.
