A quantitative approach to identify and isolate pure populations of fluorescently labeled adult retinal ganglion cells using a pressure-driven microaspiration technique.
We developed a technique to obtain pure subsets of neuronal populations for specific analysis at the molecular level. The fact that most areas of the brain consist of mixed types of neuronal and non-neuronal cells was circumvented by retrograde labeling of retinal ganglion cells (RGCs) either from the superior colliculus (SC) or the optic nerve (ON) using fluorescent dyes (4Di-10ASP or Rhodamine). Subsequent enzymatic dissociation of the retina with papain allowed to identify and collect pure populations of RGC by using the pressure-driven microaspiration technique. Enzymatic treatment and additional mechanical dissociation destroy most of the vulnerable ganglion cells leaving between 1.8% and 6% of the labeled cells intact. This low outcome was consistent among the techniques used to apply the dye and the two different dyes used in the study. However, the total numbers of cells obtained from each retina were sufficient to collect about 200 cells within 1 day and process these cells for molecular biology. As an example of further processing of collected cells, the expression of beta-actin gene was analyzed.
Hong
Y M
YM
Department of Ophthalmology, University of Tübingen, School of Medicine, Germany.
Thanos
S
S
D000818:Animals; D003584:Cytological Techniques / Q000295:instrumentation*; D005260:Female; D005456:Fluorescent Dyes; D006868:Hydrolysis; D009900:Optic Nerve / Q000166:cytology; D010206:Papain; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000502:physiology*; D012235:Rhodamines; D013396:Suction; D013477:Superior Colliculi / Q000166:cytology
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D012235:Rhodamines; C027755:rhodamine isothiocyanate; D010206:Papain