Establishment of pure neuronal and muscle precursor cell cultures from Drosophila early gastrula stage embryos.
Primary cultures of Drosophila gastrula stage embryonic cells will divide and terminally differentiate into morphologically recognizable neurons and muscles. The phenotypically mixed nature of this primary culture system has made it difficult to effectively analyze various parameters of cell growth and differentiation for individual cell types. We report here a simple and economic method to separate early embryonic precursors for different cell types, using a shallow linear reorienting Ficoll gradient at unit gravity. The separated cells were collected into fractions, cultured, and analyzed for their growth and differentiation patterns. The larger and denser cells of the first fractions differentiated to yield pure neuronal cultures, as judged by morphologic, immunologic, and biochemical criteria. Cells in the last fractions differentiated into a predominantly muscle-enriched cell population, which also contained a very small percentage of neurons morphologically distinct from those in the pure neuronal fractions. Approximately 35% of the early gastrula stage embryonic cells differentiate into neuronal cells, and 65% of the non-neuronal lineage cells later develop into predominantly muscle population. The method is highly reproducible, can process 3 x 10(7) cells per procedure, and the recovery is > 90% of the input cells. The separated cells are suitable for cell biological analyses as well as for biochemical and molecular studies of neuron and muscle precursors.
Hayashi
I
I
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Perez-Magallanes
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M
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002458:Cell Fractionation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002795:Choline O-Acetyltransferase / Q000032:analysis; D004331:Drosophila melanogaster / Q000196:embryology*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000201:enzymology; D009132:Muscles / Q000166:cytology* / Q000201:enzymology; D009474:Neurons / Q000166:cytology* / Q000201:enzymology; D010641:Phenotype; D013234:Stem Cells / Q000166:cytology* / Q000201:enzymology; D013997:Time Factors
D002795:Choline O-Acetyltransferase