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 100% purified by a novel combination of three steps: (1) Retrograde labeling by injection of the fluorescent carbocyanine tracer DiI into the optic nerve, (2) immunopanning of dissociated retinal cells with Thy1 antibodies, and (3) micro-aspiration of labeled RGCs into glass capillaries. The retina was 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 technique allowed the collection of up to 500 RGCs per capillary tube and 1500 labeled RGCs per retina. Extraction of RNA and molecular analysis by RT-PCR from 600 RGCs shows that expression of rare genes, such as those of neurotrophic factors, can be detected. This is the first description of a rapid and reliable technique for a 100% purification of RGCs with sufficient yield for molecular analysis of rare gene expression. The protocol can be modified for the purification of other cell types. The advantages and limitations of the three-step purification method are compared with previous RGC purification protocols.
Butowt
R
R
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno 89557, USA.
Jeffrey
P L
PL
von Bartheld
C S
CS
D000818:Animals; D000831:Animals, Newborn; D002232:Carbocyanines; D002469:Cell Separation / Q000379:methods*; D002642:Chick Embryo; D002645:Chickens; D004396:Coloring Agents; D005456:Fluorescent Dyes; D015870:Gene Expression; D005821:Genetic Techniques; D020933:Neurotrophin 3 / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002232:Carbocyanines; D004396:Coloring Agents; D005456:Fluorescent Dyes; D020933:Neurotrophin 3; D012313:RNA