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colony formation assay in A2780 cells


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#1 bongiwoman

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Posted 16 September 2012 - 12:46 AM

Hello, I am desparately trying to reproduce colony formation assays in A2780 (ovarian carcinoma) cells, but eventhough I am following the protocol, the results vary like crazy. One big problem is, that I oftrn don't even get nice colonies - the colony numbers are much lower than they should be and the colonies are smaller as well. Also, I don't see the phenotype I expect but rather the opposite (comparing knock-down of a gene and control). Sometimes though I do see what I want to see - but just very rarely and I can not tell why because I am not aware of having changed anything. Please help if you can! Thanks, Angelika

#2 bob1

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Posted 16 September 2012 - 02:42 PM

Protocol please - what did you do to the cells? how were the cells beforehand? How long have you had the cells up? Have you tested for mycoplasma?...

Could it be that your hypothesis is wrong?

#3 bongiwoman

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Posted 17 September 2012 - 03:05 AM

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


#4 bob1

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Posted 17 September 2012 - 01:09 PM

It could be that the siRNA treatment is affecting attachment. Do you see colonies consistently in the untreated control cells? If not, then you really have a problem - you may need to use more cells per well or try using 100% conditioned medium.

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.

#5 ascacioc

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Posted 19 September 2012 - 01:10 PM

Why not first seed 200-400 cells per well, then siRNA, then the drugs? if Bob is right and your siRNA somehow indirectly affects the attachment of the cells, it becomes more difficult to get nice colonies. Also, how does your control for the non-knocked down cells look like? Do those have nice colonies after 7 days?




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