Problem with soft agar assay

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mn303
mn303's picture
Problem with soft agar assay

Hi,
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).

I had seen some good discussion in soft agar assay in this forum in the past. Please help me out here if you guys have any thoughts/tips/tricks.

marcus muench
marcus muench's picture
 Generally the bottom layer

 Generally the bottom layer should prevent any cells from attaching.  Is there any possibility that the bottom layer had not yet hardened before you added the top layer?  

mn303
mn303's picture
The bottom layer was hard

The bottom layer was hard enough before plating the top layer for sure. What I did was I first plated the bottom agar, let the plates sit at room temp inside the hood for an hour, and then returned the plates to 37C incubator and kept overnight at 37C (since I had too many cell lines to plate). I plated bottom agar on day 1 and put layer of top agar next day. So, may be this was the source of problem? Bottom agar may be somewhat loosened by the time I plated top layer, and cells sneaked underneath and reached the plastic surface?? Any thoughts or experience sharing would be appreciated.

Lomby
Lomby's picture
 Hi there,

 Hi there,

Did you ever find a solution to this problem? because I am experiencing the exact same thing! 

I've tried plating 2mL of 0.5% Nobel agar or LMP agarose in 6 well plates and left them to set in the fridge for 30 mins before warming them to room temp for 30 mins and plating the cell layer. But after 1 week, the cells somehow make it to the bottom on the dish and start to form a monolayer. So frustrating! Any thoughts would be appreciated. Cheers