Purification of embryonic rat motoneurons by panning on a monoclonal antibody to the low-affinity NGF receptor.
Available methods for purifying motoneurons to homogeneity from rodent spinal cord involve retrograde labelling and fluorescence-activated cell sorting, making them costly and time consuming. Motoneurons are the only neurons within the 15-day embryonic rat spinal cord to express the p75 low-affinity NGF receptor and we show that monoclonal antibody 192-IgG, which binds to the extracellular domain of p75, selectively labels a sub-population of large multipolar ventral spinal cord neurons in vitro. We have developed a bench-top panning method for purifying these motoneurons using antibody 192-IgG. Approximately 10(5) cells/spinal cord are obtained in 2 h by this method; 95% of them express p75 in culture. They rapidly put out neurites on laminin substrata, and their survival is enhanced by extracts of skeletal muscle. Using the panning method in conjunction with centrifugation on a 6.8% metrizamide cushion, separate populations of large and small motoneurons were obtained, each containing more than 90% neurons staining with antibody 192. The large motoneurons had choline acetyltransferase activities/cell approximately 4-fold greater than those of dissociated total spinal cells and 7-fold higher than those of the small motoneurons. These methods should be of considerable use for studies on factors affecting motoneuron survival and development and for transplantation of highly purified neuronal populations.
Camu
W
W
Centre de Recherche de Biochimie Macromoléculaire, UPR 8402 du CNRS/INSERM U249, Montpellier, France.
Henderson
C E
CE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002470:Cell Survival / Q000502:physiology; D002499:Centrifugation, Density Gradient; D002795:Choline O-Acetyltransferase / Q000032:analysis; D003470:Culture Media; D004622:Embryo, Mammalian / Q000166:cytology / Q000276:immunology; D005455:Fluorescent Antibody Technique; D007074:Immunoglobulin G / Q000276:immunology; D008793:Metrizamide; D051379:Mice; D009046:Motor Neurons / Q000201:enzymology / Q000276:immunology / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017475:Receptors, Nerve Growth Factor / Q000276:immunology / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007074:Immunoglobulin G; D017475:Receptors, Nerve Growth Factor; D002795:Choline O-Acetyltransferase; D008793:Metrizamide