Purification of chick retinal ganglion cells for molecular analysis: combining retrograde labeling and immunopanning yields 100% purity

Retinal ganglion cells (RGCs) from embryonic and posthatch chickens were 10 0% purified by a novel combination of three steps: (1) Retrograde labeling by injection of the fluorescent carbocyanine tracer DiI into the optic nerv e, (2) immunopanning of dissociated retinal cells with Thy1 antibodies, and (3) micro-aspiration of labeled RGCs into glass capillaries. The retina wa s dissected and dissociated with trypsin 12-15 h after the injection of DiI . DiI-labeled cells were identified on immunopanned dishes by fluorescence and collected for molecular analysis within 3 h after dissociation. This te chnique allowed the collection of up to 500 RGCs per capillary tube and 150 0 labeled RGCs per retina. Extraction of RNA and molecular analysis by RT-P CR from 600 RGCs shows that expression of rare genes, such as those of neur otrophic factors, can be detected. This is the first description of a rapid and reliable technique for a 100% purification of RGCs with sufficient yie ld for molecular analysis of rare gene expression. The protocol can be modi fied for the purification of other cell types. The advantages and limitatio ns of the three-step purification method are compared with previous RGC pur ification protocols. (C) 2000 Elsevier Science B.V. All rights reserved.