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Problem with soft agar assay


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#1 Manish Neupane

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Posted 29 January 2012 - 02:27 PM

I've done soft agar assay for several times in past in various formats. For the first time, I am doing it in 6-well plate format (by titrating everything down based on the surface area differences between different plates).

However, now I came across a bizzare problem. I can see many cells adhered to the plate within a few days of cell plating and growing in monolayer instead of forming typical three-dimensional colonies. I know I had no problem in pouring the agar. These are breast cancer cells. Bottom layer is 2.5 ml of 0.5% Agar Noble (in DMEM) per well and top agar is 1 ml of 0.33% Agar Noble per well (with 25,000 to 100,000 cells in MEGM).

Somebody in this forum had posted similar problem in August 2010, which got no attention or reply from anybody. Please help me out here if you guys have any thoughts.

Manish

#2 klinmed

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Posted 31 January 2012 - 11:41 AM

Changing the format may increase the time the bottom agar needs to FULLY set. Would suggest you try chilling the bottom layer at 4oC before plating the top layer. If problems persists maybe try a slight increase in agar concentration of the bottom layer (0.7%?).

#3 Manish Neupane

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Posted 31 January 2012 - 12:49 PM

Thanks for the reply. I left the bottom agar layer at room temp for an hour and then put the plates in 37C incubator overnight before putting top layer. So, probably they were set well-but may be putting in incubator overnight before plating top layer was a mistake?




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