Protocol for Making Stable Cell Lines

1. Plate 2 x 106 S2 cells in each well of a six well tissue culture dish.
2. Incubate at 25°C for 4-5 hours.
3. Transfect with 2 µg total DNA (1µg expression vector + 1µg selection vector) + 16 µL Enhancer (Qiagen Effectene kit) + 182 µL EC buffer (Qiagen Effectene kit). Vortex, briefly spin down and incubate at RT for 5'. Add 20 µL of Effectene. Vortex, briefly spin down and incubate at RT for 10'-15'.
4. Add Transfection reaction to cells drop-wise.
5. Incubate for 3d and split at fairly dense dilution to two wells, and add selection medium (usually containing Hygromycin at 150-200 µg/mL) for 3d. After that, it is a careful balance of selection and switching to WT medium (without Hygromycin) and seeing how the cells look from day to day. Ideally they are a little sick looking as they get selected, but still dense. Keep one well held over from the last time they looked good in case the real selected one crashes. After they prove they can grow and be split at least once in selection medium, immediately freeze an aliquot of this partially selected cell pool in case they all die. Assuming they do stay alive and keep growing, freeze down real stocks after a few more passages in the selection medium. The result is a mixed population stable cell line (this is different from a clonally isolated cell line).

Having trouble? Here are a few tips.

1. Try again. Sometimes things just don't go well with one round but on another attempt, they do.
2. If you're trying to express a protein that might be toxic to cells, try an inducible promotor, so you can recover cells with the plasmid independent of expressing the protein.
3. Check your DNA or re-prep it. A good clean midi prep is recommended. If the DNA is not clean enough or precipitates, that can be a problem (e.g. kill the cells).
4. Take a look at manufacturers' protocol handbooks. For example, Invitrogen has a handbook of information about their S2 cell lines that might be helpful.
5. Check out this paper:

   Santos et al. (2007) Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression. Cytotechnology (2007) 54:15-24. DOI 10.1007/s10616-007-9060-9 ()