For now i've tried in 6 well plates coated at 0.6 % agar (1,5ml) and cells at 3 different concentration (20 000, 50 000 and 100 000 cells per well) in 0.4% agar (1,5 ml). I used media 2x + 20% FBS so the cells are happy in media +10% FBS final (as they normally grows in). I got 5-10 colonies after 14 days in the 20 000 cells. I've stained with crystal violet 0,05% for 1h and wash a bit with water but that wash doesn't seem to matter.
I've tried again but this time in 96 well plate as already published eith those cells. I've used the same concentration of agar and put 100ul of coating agar and 100ul of cells in agar (2000 cells per well). and incubated for 11 days and got nothing.
When I'm aspirating a part of the media or the stain do I aspirate the colonies? I'm far from touching the agar layer thought. I'm using selec agar from sigma, should I use another type of agar? Basically I don't know what i'm doing good or wrong here. Tks for your help.
Your procedures are unclear. Could you give a brief overview of your purpose and problem? "I've stained with crystal violet 0,05% for 1h and wash a bit with water but that wash doesn't seem to matter" Does this mean that crystal violet did not stain or water did not destain or there were no cells there?
Do you mean your cultures did not work in 96 well plates? These are tricky, you seem to have gone from 6 to 96 wells. Perhaps you can try 24 wells, those work well for 3D.
Hope this helps
Hello
I agree with Victoria M Virador , we need to explain more to help you
please , look at link here may help you
Good Luck
http://clincancerres.aacrjournals.org/content/15/12/4038.full
For the staining, when i had some colonies, the destain did not seem to wash at all the stain and seems to not be needed.
I first started in 6 well-plates since it was what have been tried before in our lab but unsuccessfully. When I passed upon a paper that did it in 96 well plate with the cell line that I'm using, I've decided to give it a try. Maybe an in between would be good.
Thanks for the link I'll read it and see if it helps.
Do you have any advice on what I should avoid while seeding the cells and how to change media and do treatment without disturbing the cells, can I use suction to remove media? How often do you change media on top of the agar?
Thanks again for your help
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