The following protocol was developed by Katherine Sepp and Jianwu Bai during their postdoctoral work in the Perrimon laboratory at Harvard Medical School.
Consideration before starting: What genotype?
For primary cell screening, researchers typically construct a fly line in which the lineage of interest (for example, neuronal or muscle cells) is tagged with GFP. That makes it possible to identify the cells of interest (such as during a subsequent fluorescence microscopy-based screening assay) from the mixed population of many cell types that will be present in the prep. The appropriate genotype or cross should be constructed and expanded (a large number of flies are needed to obtain a sufficient volume of embryos).
Protocol for primary cell preparation from an appropriate genotype
You will need:
- Fly cages and molasses plates
- Dechorionation baskets
- a Dounce homogenizer
- access to a tissue culture hood
- a clinical centrifuge
- culture media
- Maintain flies at 25°C in large culture cages, feeding them killed yeast paste streaked on molasses plates.
- On tissue culture day, do a one-hour pre-lay with fresh food. Then, provide another fresh plate (the collection plate) and collect eggs for 2 h. Remove the plate and incubate the embryos on the collection plate for a further 4 h.
- Wash the embryos with water and place into a standard dechorionation basket.
- Dechorionate embryos in 50% household bleach for 5 min. Proceed to the tissue culture hood, where the rest of the prep is completed.
- Rinse the embryos with sterile distilled water, then immerse in Drosophila media (i.e. Schneider's).
- Blot the embryos dry and transfer to Dounce homogenizers filled with the appropriate amount of Drosophila media (the appropriate volume will depend on the size of the vessel).
- Gently but firmly Dounce to the bottom, using 7 to 15 strokes.
- Transfer the homogenate into conical centrifuge tubes and spin at 40 x g in a clinical centrifuge for 10 min.
- Transfer the supernatant very carefully and spin at 380 x g for 10 min to bring down the cells.
- Discard the supernatant. Resuspend the cell pellet in primary culture cell medium (10% Fetal Bovine Serum, 10 U/mL penicillin, 10 µg/mL streptomycin in Schneider's/Shields and Sang medium).
- Draw 40 µL of cell suspension and mix well with 8 µL of 0.4% trypan blue solution. Stain for 5 min. and count on a hemocytometer for cell density and viability.
- Dilute the cell prep to the desired concentration. To start, try a range of 1-5 x 106 cells/mL and plate out at 1.7 to 2.5 x 105 cells/cm2.