Flow cytometry for quantification of retrogradely labeled retinal ganglion cells by Fluoro-Gold

Purpose. To count retrogradely labeled retinal ganglion cells by Fluoro-Gold. Methods. Retinal ganglion cells were retrogradely labeled using bilateral injections of Fluoro-Gold into the superior colliculus. One week after injections, retinas were dissociated and immunolabeled using specific antibody against Fluoro-Gold. The Fluoro-Gold labeled cells were then counted using flow cytometry. Results. Flow cytometry revealed that approximately 7% of the dissociated retinal cells were ganglion cells retrogradely labeled by Fluoro-Gold (Fig. 1B). Based on the total count of retinal cells per eye, the total number of retinal ganglion cells was estimated at approximately 131,250 ± 2,542 per rat eye. The coefficient of variation of counts was calculated as 1.98%. Conclusions. The use of flow cytometry facilitates simple, reproducible and rapid quantification of virtually all of the retinal ganglion cells labeled by Fluoro-Gold in a single rat eye.