Combined methods of retrograde staining, layer-separation and viscoelastic cell stabilization to isolate retinal ganglion cells in adult rats.
The adult retinal ganglion cells (RGCs) are widely used as a model to study mechanisms of de- and regeneration within the central nervous system (CNS). All regions of the CNS including the retina have the common disadvantage of being composed of heterogeneous populations of cells, many of them like RGCs making less than 1% of the total population. This disadvantage can be circumvented by methodologies aimed at purifying specific cell types. Here we describe a method that combines retrograde labelling with fluorescent dyes and the pull-off technique. By using either of four different fluorescent dyes to retrogradely label RGCs, between 55 and 80% of pre-labelled ganglion cells could be identified with fluorescence microscopy. The pull-off procedure was supplemented by enhancing the viscosity of the collecting medium with methylhydroxypropyl-cellulose (MHPC). It appeared that 45% of all RGCs could be collected in the medium containing MHPC as compared to about 19% of RGCs which could be collected in medium devoid of cellulose. Despite the fact that morphometric measurements indicated that 60% of the cells collected fulfilled the criteria of being RGCs, the population obtained was immensely enriched and sufficed to perform a two dimensional SDS-gel electrophoresis and to determine their protein composition. The collected RGCs displayed a similar protein pattern as compared to that of the total retina, indicating that sublayers of the retina were represented in the probe. The results suggest that combined approaches of in vivo staining and ex vivo separation within suitable media enables the collection of an enriched population of cells which can be processed for biochemical analysis, and perhaps for molecular biology.
Department of Experimental Ophthalmology, School of Medicine, University of Münster, Germany.
P
P
Simon
S
S
Thanos
D000367:Age Factors; D000818:Animals; D002469:Cell Separation / Q000379:methods*; D004548:Elasticity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D065347:Hypromellose Derivatives; D008747:Methylcellulose / Q000031:analogs & derivatives; D011480:Protease Inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000166:cytology*; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling / Q000379:methods*; D013266:Stilbamidines; D013501:Surface-Active Agents
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D013266:Stilbamidines; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D065347:Hypromellose Derivatives; D008747:Methylcellulose