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

被引:17
作者
Butowt, R
Jeffrey, PL
von Bartheld, CS
机构
[1] Univ Nevada, Sch Med, Dept Physiol & Cell Biol, Reno, NV 89557 USA
[2] Childrens Med Res Inst, Wentworthville, NSW 2145, Australia
关键词
panning purification; DiI fluorescent label; retina; micro-aspiration; Thy; 1; antibody; RT-PCR;
D O I
10.1016/S0165-0270(99)00149-1
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2000 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:29 / 38
页数:10
相关论文
共 38 条
[1]  
Aguayo AJ, 1996, CIBA F SYMP, V196, P135
[2]   NEONATAL RETINAL GANGLION-CELL CULTURES OF HIGH-PURITY - EFFECT OF SUPERIOR COLLICULUS ON THEIR SURVIVAL [J].
ARMSON, PF ;
BENNETT, MR .
NEUROSCIENCE LETTERS, 1983, 38 (02) :181-186
[3]  
AUSTIN CP, 1995, DEVELOPMENT, V121, P3637
[4]   IMMUNOLOGICAL, MORPHOLOGICAL, AND ELECTROPHYSIOLOGICAL VARIATION AMONG RETINAL GANGLION-CELLS PURIFIED BY PANNING [J].
BARRES, BA ;
SILVERSTEIN, BE ;
COREY, DP ;
CHUN, LLY .
NEURON, 1988, 1 (09) :791-803
[5]  
BRAY GM, 1991, ANN NY ACAD SCI, V633, P214
[6]   Survival and differentiation of purified embryonic chick retinal ganglion cells cultured at low density in a chemically defined medium [J].
Brocco, MA ;
Panzetta, P .
JOURNAL OF NEUROSCIENCE METHODS, 1997, 75 (01) :15-20
[7]   Highly sensitive method to detect mRNAs in individual cells by direct RT-PCR using Tth DNA polymerase [J].
Chiocchia, G ;
Smith, KA .
BIOTECHNIQUES, 1997, 22 (02) :312-&
[9]   Retinal cultures [J].
Finlay, D ;
Wilkinson, G ;
Kypta, R ;
deCurtis, I ;
Reichardt, L .
METHODS IN CELL BIOLOGY, VOL 51, 1996, 51 :265-283
[10]   PARTIAL CHARACTERIZATION OF CHICKEN THY-1 GLYCOPROTEIN BY MONOCLONAL-ANTIBODIES [J].
FRENCH, PW ;
JEFFREY, PL .
JOURNAL OF NEUROSCIENCE RESEARCH, 1986, 16 (03) :479-489