colony formation assay in A2780 cells
Posted 16 September 2012 - 12:46 AM
Posted 16 September 2012 - 02:42 PM
Could it be that your hypothesis is wrong?
Posted 17 September 2012 - 03:05 AM
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.
Posted 17 September 2012 - 01:09 PM
From the pictures I found, it looks like the cells are quite small, and have a tendency to grow in clumps too. This may mean that they don't do well as a single cell suspension, as it would indicate that they like to have lots of "friends" around. If this is the case, you may need to go to a feeder layer or something similar.
You could try 20% FCS to help the cells survive the double treatment.
Posted 19 September 2012 - 01:10 PM