Department of Development and Cell Biology, Department of Anatomy and Neurobiology, University of California, Irvine (UCI)
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Sicaeros, B., O'Dowd, D. K. Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos. J. Vis. Exp. (5), e226, doi:10.3791/226 (2007).
This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24°C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.
I. Drosophila Embryo Collection
II. Embryo Dechorionation with Bleach
III. Preparation of single embryo cultures
IV. DDM1 Defined medium for growing the cultures
In Drosophila cultures prepared from midgastrula stage embryos the neurons arise from neuroblast precursors, many of which divide prior to differentiation in vitro. This system thus provides a unique opportunity for exploration of genetic and environmental factors important in very early phases of neuronal development (Rohrbaugh et al., 2003). We have shown that neurons grown in a simple defined medium differentiate into electrically excitable neurons that also form functional synaptic connections (O Dowd, 1995; Lee and O Dowd, 1999). Using analysis of mutants and/or pharmacological manipulations, in combination with standard whole cell recording techniques this model system can be used to explore the role genes and environmental factors involved in development of electrical excitability and synaptic transmission (Hodges et al., 2002; Lee and O Dowd, 2000; Lee et al., 2003).
This work was supported by NIH grant NS27501 to DKOD. Additional support for this work from an HHMI Professor Grant to DKOD.
|Drosophila melanogaster||Animal||Fruit flies|
|Transferrin||Reagent||Sigma-Aldrich||T-1147||100x Stock: 10 mg/ml in water. Filter through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20C.|
|Insulin||Sigma-Aldrich||I-6634||200x Stock: 10 mg/ml in 0.05N HCl. Filter through 0.2 um syringe filter (cellulose acetate). Store in 120 ul aliquots (for 20 ml DMEM) at -20C.|
|Putrescine||Sigma-Aldrich||P-5780||100x Stock: 10 mM in ddH2O. Filter through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20 C|
|Selenium||Sigma-Aldrich||S-5261||100x Stock: 3 uM in ddH2O. Put 0.0051 g Selenium in a 15 ml tube labeled A (3 mM stock). Add 10 ml of sterile water. Take 10 ul from Tube A to Tube B with 10 ml ddH2O (3 uM stock). Filter Tube B through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20C|
|Progesterone||Sigma-Aldrich||P-6149||100x Stock: 2 ug/ml1.Add 1ml of 100% EtOH to 0.001 g Progesterone bottle2.Add 49 ml ddH2O3.Transfer 1 ml of this to second tube with 9 ml ddH2O4.Filter through 0.2 um syringe filter (cellulose acetate)5.Store in 220 ul aliquots (for 20 ml DMEM) at -20C|
|DDM1||Medium||To 10 mls of DMEM add from stocks:100 ul Transferrin, 100 ul Putrescine,100 ul Selenium,100 ul Progesterone, 50 ul Insulin|
|Cover-slips||Bellco Glass||1943-00012||Low lead glass, autoclaved.|
|Paper filter||Whatman, GE Healthcare||#1|
The cultures made in this media must be maintained in a 5% CO2 incubator at about 22-24°C. We use a standard mammalian tissue culture incubator placed in a cold room and heated to 23°C. Always used autoclaved filtered water and make in containers that are reserved for media and supplements only. Never put a pH meter or stir bar used for other purposes in media.
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