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.
bongiwomanMember Since 16 Sep 2012
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16 Sep 2012 - 12:52