Flow cytometry for quantification of retrogradely labeled retinal ganglion cells by Fluoro-Gold.
OBJECTIVE
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.
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J
J
Yang
G
G
Tezel
R V
RV
Patil
M B
MB
Wax
D000818:Animals; D002452:Cell Count; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012165:Retinal Ganglion Cells / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods; D013266:Stilbamidines
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D005456:Fluorescent Dyes; D013266:Stilbamidines