[zoekelly (OP)]

Hi

Does anyone have any good explanations (ideally with references!!) as to why melanoma cell lines would not form colonies in a standard clonogenic assay (no drugs, just seeding cells) or in a soft agar assay? Even any information about other cancer cell lines would be helpful. The cells I used did not form colonies and i need an explanation for my thesis.

Thanks

Zoe

[chris]

I have limited experience in working with cancer cell lines, but of those that I have used there is the usual 'critical density' phenomenon whereby if the cells are too widely dissociated and make too few contacts with other cells then they will not grow. One cell line I'm using at the moment does precisely this. When you seed a flask with them the cells only grow around the edges of the flask where their numbers are sufficiently high.

Do please let me know if you arrive at a more satisfactory conclusion.

chris

"I never forget a face, but in your case I'll make an exception"
 - Groucho Marx

[MayoFlyFarmer]

I'm with chris that with cell culture it is quite likely something with your technique, rather than something inherant to the cells that doesn't let them grow.  Although cancer cells are usually pretty easy (hence why they are a bad thing in vivo). How many cells are you starting out with? what kind of medium are you using? etc. sometimes its just a metter of playing around with the variables.

We don't want the loonies taking over!

[zoekelly (OP)]

Hi

Thanks for the replies. This experiment was done at least 4 times in triplicate each time. I plated densities from 500 cells up to 2000 and even at 2000 cells per 6cm plate, no colonies formed after 3 weeks. This was done in 2 different laboratories. Most cancer cell lines will form colonies at densities of as low as 100 cells per 6cm dish. The medium i used was the same as the medium used to grow the cells in, but maybe i needed to enrich it. At this stage i'm nearly finished writing up so there is no chance of doing it again. But i am looking for potential reasons why?
Thanks
Zoe

[MayoFlyFarmer]

when you say they didn't form colonies, do you mean they wouldn't grow period?  or wouldn't generate foci??

A submarine is NOT a cargo ship!

[MayoFlyFarmer]

also, if you can, look up the actual type of medium you used.  I'm just curious.


A submarine is NOT a cargo ship!

[zoekelly (OP)]

Hi
When i say they didn't form colonies, after 2/3 weeks there were floating things in my petri dishes (definitely TCP plates) and nothing ahd stuck, nothing had grew, nothing at all. I plated like 10000 cells and they stuck grand but any low densities normal for colony formation just wouldn't stick.
The medium was DMEM with Glutamax, 10% FBS and 4ug/ml of insulin. They grew absolutely fine in this medium normally. Even when i tried a soft agar assay which has somethign ridiculous like 20% serum in it absolutely nothing happened.

Any suggestions would be great.

Thank you so much
Zoe

[MayoFlyFarmer]

did you have any anti-biotics in your medium?? (pen/strep, etc.?)

A submarine is NOT a cargo ship!

[zoekelly (OP)]

Hi
The first 3 times i did it yes i had antibiotics in my medium. The 4th time i did it, in a different lab with someone checking me - all experts in doing this kind of thing, there were no antibiotics in the medium. Same medium though.
Zoe