Culturing S2 Cells
Introduction The S2 cell line was derived from a primary culture of late stage (20-24 hours old)
Drosophila melanogaster embryos (Schneider, 1972). Many features of the S2 cell line
suggest that it is derived from a macrophage-like lineage. S2 cells grow at room
temperature without CO2 as a loose, semi-adherent monolayer in tissue culture flasks and
in suspension in spinners and shake flasks.
General Cell
Handling
General guidelines are provided below to help you grow S2 cells.
? All solutions and equipment that come in contact with the cells must be sterile.
? Always use proper sterile technique in a laminar flow hood.
? All incubations are performed in a
you want to slow down S2 cell growth, you may incubate cells at room temperature
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? The complete medium for S2 cells is Schneider.s Drosophila Medium containing
10% heat-inactivated FBS. This medium is used for transient expression and stable
selection. Schneider.s Drosophila Medium is available separately from Invitrogen
(Catalog no. 11720-034). Heat-inactivated FBS must be added to a final
concentration of 10% before use.
? Optional: Use Penicillin-Streptomycin at a final concentration of 50 units penicillin
G and 50 ìg streptomycin sulfate per milliliter of medium.
? Before starting experiments, be sure to have established frozen S2 cell stocks.
? Count cells before seeding for transfection or freezing cells for stocks. Check for
viability using trypan blue. S2 cell viability in culture should be 95-99%.
? Always use new flasks or plates when passing cells for general maintenance. Duringtransfection and selection keep cells in the same culture vessel.
? For general maintenance of cells, pass S2 cells when cell density is between
6 to 20 x 106 cells/ml and split at a 1:2 to 1:5 dilution. Note: S2 cells do not grow
well when seeded at a density below 5 x 105 cells/ml.
For example, transfer 2 ml of a 10 ml cell suspension at 2.0 x 107 cells/ml to a new
? S2 cells grow better if some conditioned medium is brought along when passaging
cells. Note: Conditioned medium is medium in which cells have been grown.
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S2 cells do not completely adhere to surfaces, making it difficult to rinse the cells if
needed. To exchange cells into new medium or to wash cells prior to lysis, follow the
instructions below:
? Resuspend cells in the conditioned medium and centrifuge at 1000 x g for 2 to 3
minutes. Decant the medium.
? Resuspend the cells in fresh medium (or PBS) and centrifuge as above. Repeat.
? Add fresh medium (or buffer) and replate the cells (or lyse them).
