bongiwoman

ok, so here is the protocol:
1) Day 1 seed 150.000 cells per well in a 6 well plate
2) Day 2 Transfect with control siRNA and target siRNA using Lipofectamine (a very standard protocol that everybody uses; Western Blot shows that knockdown worked very well)
3) Day 3 Trypsinize cells and seed 200 or 400 cells per well in 2ml medium (6 well plates)
4) Day 4 Treat with several cytotoxic drugs and leave one plate of each (ctr and knock down) as untreated control
5) After 2 hours, remove medium and replenish with fresh medium (have tried normal as well as 50% conditioned)
6) After 7 days, stain colonies with crystal violet solution.

The cells were at an early passage number, like 6 or so - I thawed new ones after the old passage number had stopped working, but it did not help.

Before the experiment, I did not do anything special, just 2 times splitting per week - I also tried several ratios to make sure they were fine.

Mycoplasma - not tested yet but pretty sure this is not the problem. The cells in the flasks look all fine, but the assay just does not work.

Any clue....?

Thanks,

Angelika