Primary Culture of Drosophila Embryo Cells
Cultures are initiated from embryos in the early gastrula stage, making it possible to observe cell development from committed precursor to final differentiated cell type. The types of cells that differentiate in culture are primarily neurons (Seecof et al., 1973a) and multinucleate myotubes (Seecof et al., 1973b), but other epidermal and mesodermal derivatives are also present (Shields et al., 1975). Cultures form a variety of neurons with highly differentiated phenotypes, including neurotransmitter systems (Salvaterra et al., 1987), ion channels (Byerly and Leung, 1988; Germeraad et al., 1992), axonal specializations, and even functional neuromuscular junctions (Seecof et al., 1972) and synaptic activity (Lee and O'Dowd, 1999). Medium conditioned by embryo cells has also been shown to contain a variety of activities that can modulate the growth and differentiation properties of neurons (Salvaterra et al., 1987; Hayashi et al., 1992). Figure 3 shows some of the cell types observed in a differentiated Drosophila primary embryo culture and neurons marked with a fluorescent transgenes specific for different neurotransmitter phenotypes. Remarkably, cultured Drosophila embryo cells have also been shown to express a temporally specific transcriptional factor cascade thought to be important for establishing the developmental potential of subsequent cell lineages from primary neuroblasts (Brody and Odenwald, 2000, 2002).
Penicillin and streptomycin are from GIBCO/BRL (Cat. No. 15140-122). Insulin is from Sigma (Cat. No. 1- 5500). 1X Schneider's Drosophila medium with glutamine is from GIBCO (Cat. No. 11720-034). Fetal bovine serum is from GIBCO (Cat. No. 10082-139).
Cells are usually cultured in 35-mm plastic dishes from Corning (Cat. No. 430165). For finer morphological observations, cells are plated directly onto round glass coverslips placed in the bottom of a 35-mm plate (five per culture dish, Fisher Scientific, Cat. No. 12-545- 801-1THK). Coverslips are presterilized using standard gas or autoclave procedures.
Dechorionated embryos are dissociated in 15-ml Dounce glass-glass homogenizers from Baxter Diagnostics (Cat. No. T4018-15). Nylon mesh (25-100 µm pour size) can be obtained from most fabric stores. All other equipment and reagents are commonly used for general laboratory work or tissue culture and can be obtained from any scientific supply company.
A. Egg Collection and Aging to Early Gastrula Stage
Yeast-proprionic acid paste: Dissolve dry yeast in 2% proprionic acid
Grape-juice-agar egg collection plates: 160ml water, 160ml grape juice (we use Welch's), 6.4 g Bacto-agar (Becton, Dickinson, Cat. No. 214010), 0.8g Nipagin [p-hydroxybenzoic acid methyl ester (methylpraben)] (Sigma, Cat. No. H-5501). Combine water, grape juice, and Bacto-agar in a l-liter flask. Microwave to dissolve agar, being careful not to let the solution boil over. Remove from the microwave, add Nipagin, mix, and pour into 12 large (14cm) culture plates.
B. Harvesting of Embryos
Deionized water: Prepare 2 to 3 liters of deionized water.
C. Preparation of Embryonic Cultures
Penicillin-streptomycin stock solution: Penicillin-streptomycin solution (10,000U/ml) is obtained from GIBCO/BRL (Cat. No. 15140-122). Use 10ml per liter of medium (i.e., final concentration of 100 U / ml penicillin, 100 µg/ml streptomycin).
Insulin stock solution: Dissolve insulin in 0.05 N HCl at a final concentration of 2mg/ml. Filter sterilize (0.2 µm) and store at 4°C (stock is stable for 1 year). Use at a final concentration of 0.2 µg/ml of medium.
Schneider's Drosophila medium with glutamine, insulin, and 5% fetal bovine serum: Add 10ml of sterile penicillin/ streptomycin stock solution, 0.1 ml of insulin stock solution, and 50 ml of fetal bovine serum (final serum concentration = 5%) to 1 liter of Schneider's Drosophila medium with glutamine. The medium supports good differentiation when stored at 4°C for up to 1 month.
Sodium hypochlorite-ethanol: In a small beaker mix 10 ml of 95% EtOH and 10ml of 3% sodium hypochlorite. This solution will dechorionate the embryos and sterilize them.
Deionized water: Prepare about 1 liter of sterile deionized water.
Cells are usually grown in 35-mm dishes but it is also possible to plate directly onto coverslips (see earlier) and even to grow cells in the raised condensation rings found on the lids of many 96-well microtiter plates. This is especially convenient when screening monoclonal antibodies, as each embryo culture can be isolated and matched with the corresponding position for a hybridoma clone growing in a 96-well culture dish. A proceedure has been described for treating the lids of culture plates with H2SO4 that results in a significant improvement in the attachment and differentiation of Drosophila embryo cells (Furst and Mahowald, 1984).
The yield of cells depends on how vigorously the cells are dissociated and how long the embryos are exposed to the ethanol-hypochlorite mixture. Using the previously described procedures, we can routinely obtain ~106 cells for plating from a single embryo collection bottle stocked with about 3000 adult flies. This is sufficient for preparing five 35-mm culture dishes. The procedure can be scaled up or down easily using additional embryo collection bottles or using a smaller egg-laying population.
In the past we have used up to 20% fetal calf serum; however, such high concentrations are not necessary to support optimal growth and differentiation. It is especially important to use prescreened lots of fetal calf serum that are suitable for Drosophila embryo cultures. Some lots of fetal calf serum do not support differentiation and even appear to be toxic to Drosophila embryonic cells. We have had good success with serum from GIBCO and Gemini Products. A chemically defined medium for preparing embryo cultures has also been described (O'Dowd, 1995). The surface coating of tissue culture plasticware varies from different manufacturers. We have found that some types of 35-mm plastic dishes, such as those from Falcon, are not optimal for Drosophila cultures.
Work in the authors' laboratories has been supported by grants from NIH-NINDS. We thank our past and current laboratory colleagues for many helpful suggestions and ideas about Drosophila primary embryo cultures, especially Drs. N. Bournias- Vardiabasis and S. Song.
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