Primary Culture of Drosophila
|FIGURE 1 Large plastic jars used for embryo collections. The jar
on the left is empty and the one on the
right has a grape juice agar
embryo collection plate taped to the bottom and is stocked with
about 3000 adult
flies as described. Holes have been cut into the
sides of the jars and are covered with nylon mesh to allow
Primary cultures of Drosophila
embryonic cells offer a
unique system to study the transitions of undifferentiated
cells into a variety of cell types. Coupled with the
power of Drosophila
classical and molecular genetics,
the analyses that are possible in vitro using differentiating
embryo cells will continue to contribute to a deeper
understanding of development. Preparation of primary
embryo cell cultures was developed independently in
the laboratories of Seecof and colleagues (Seecof and
Unanue, 1968) and Shields and Sang (1970). The procedures
for preparing cultures are technically undemanding
and can be adapted to solve a wide range of
biological problems. Important variations include the
culture of single embryos (Cross and Sang, 1978), as
well as the development of techniques for isolating the
precursors of different cell types such as neurons and
muscle cells (Furst and Mahowald, 1985; Hayashi and
Perez-Magallanes, 1994). Figure 1 shows a convenient
way to collect embryos from large populations of egglaying
adults, and Fig. 2 illustrates a typical setup used
for preparing primary embryo cultures.
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).
II. MATERIALS AND
|FIGURE 2 Typical items used to prepare Drosophila gastrula
stage embryo cultures are arranged in a small
laminar flow hood.
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
Solution and Egg Collection Plates
Yeast-proprionic acid paste
|FIGURE 3 A Drosophila gastrula stage embryo cell culture after
about 2 days of in vitro differentiation. The
majority of cells
differentiate into neurons organized in clusters. This image is a
composite of a bright-field
image overlaid with a false-colored
fluorescent image of the same field. Green cells are neurons
a cholinergic-specific fluorescent transgene and are
indicative of cells with a cholinergic neurotransmitter
White arrowheads show some multinuicleate myotubes, white and
the asterisk indicates axonallike
processes emanating from neurons
or neural clusters and are often connected to neurons in adjacent
Scale bar: 50 µm.
: Dissolve dry yeast in 2%
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
-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)
B. Harvesting of Embryos
- Prepare large egg-laying population by transferring
1.5-week-old adult flies from 8 to 12 ordinary
culture bottles to fresh culture bottles supplemented
with dry yeast sprinkled over the food. Allow the
population to adapt for 1-2 days in a 25°C humidified
room and transfer the adults to an embryo collection
bottle on the day cultures will be prepared. Figure 1
shows an example of a homemade collection bottle
or a similar one can be obtained commercially (Doc
Frugal Scientific, Cat. No. 55-100). We generally stock
each egg-laying population with about 3000 adult flies.
For convenience, maintain the room on a reverse
light-dark cycle so collections can be done in the investigators'
morning (i.e., the flies' dusk).
- Apply a small amount of yeast-proprionic acid
paste on a small piece of filter paper and place in the
center of a large (14-cm) grape juice-agar egg collection
plate to attract females to lay eggs.
- Invert the embryo collection bottles onto the food
plate and collect eggs for 1 or 2h.
- If better developmental timing (synchronization)
is desired, do a 30-min to 1-h precollection and discard
plates. This may be necessary to induce females to lay
eggs they have been storing for longer times.
- Remove egg collection plates from the embryo
collection bottles, cover, and allow embryos to develop
to early gastrula stage at 25°C for 3.5 h.
Prepare 2 to 3 liters of deionized water.
- Embryos should be harvested 5-10min before
the 3.5-h aging period so the cultures can be initiated
immediately at 3.5 h.
- Discard filter papers with yeast mixture.
- Transfer plates to sink and rinse extensively and
gently with deionized water to remove adult flies and
- Pour water on the plates and gently loosen
embryos with a camels' hair paint brush.
- Pour suspended embryos into a nylon mesh filter
to allow further washing and collection from other
- Continue washing and remove large food particles
and other debris with forceps if necessary.
- Transfer nylon mesh with embryos to a sterile
tissue culture hood.
C. Preparation of Embryonic Cultures
Penicillin-streptomycin stock solution
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.
: In a small beaker mix 10 ml
of 95% EtOH and 10ml of 3% sodium hypochlorite.
This solution will dechorionate the embryos and
: Prepare about 1 liter of sterile deionized
- Preclean the laminar flow hood with ethanol
using normal tissue culture procedures. All further
operations are carried out under sterile conditions in
- Transfer embryos to the beaker of sodium
hypochlorite-ethanol using a sterile spatula and swirl
gently for 1 to 2min. The time may vary slightly but
do not exceed 3 min. Timing depends on the freshness
of the hypochlorite and the compromises you are
willing to make with respect to yield and sterility.
Shorter times tend toward better yields and longer times are necessary if you wish to totally eliminate
- Pour dechorionated embryos onto a small nylon
mesh filter funnel and wash extensively with about
500ml sterile distilled water.
- Transfer to a 15-ml Dounce homogenizer that
contains about 6 ml of culture medium and dissociate
cells by gently homogenizing with three rotary up and
- Filter dissociated cells through a 25- to 100-µm
nylon mesh filter (the mesh is taped loosely over a 5-
cm-diameter glass funnel or fitted into a 3.5-cm metal
ring that is placed into the funnel) to remove undissociated
clumps and other large debris.
- Collect filtrate in a 15-ml centrifuge tube and
pellet cells by centrifugation at 1400rpm for 4min.
Wash cells one time in 6 ml of culture medium and centrifuge
as described earlier, followed by a final suspension
in 10 ml of culture medium.
- Place a small aliquot in a hemocytometer and
count the round, intact, medium-sized cells and cell
clusters to determine cell number.
- Calculate the concentration of cells, add sufficient
medium to obtain a final density of 8 × 105 cells/ml, and plate approximately 1.6 × 106 cells in a
35-mm culture dish (i.e., 2ml of cell suspension per
- Culture cells in a humidified 25°C incubator (CO2 is not necessary). Cells begin to differentiate in a few
hours and are substantially developed after about
24h. Cells can be cultured for several days before
replacing the medium. It is possible to keep cultures
for more than 1 year.
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 H2
that results in a
significant improvement in the attachment and differentiation
of Drosophila embryo cells (Furst and
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
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
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
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
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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