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Question regarding soft agar assay.


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#1 crom80

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Posted 25 July 2013 - 07:42 AM

I am trying to perform a soft agar assay using the protocol found here:

http://web.mnstate.e...ay Protocol.pdf

The major difference with the given protocol and mine is that I use 20% FBS media instead of 2X RPMI. Also, the protocol instructs to use an agarose based cell layer, and I have read that agarose is better than agar. Also, does the agar make a difference? I use a generic agar from millipore and not LB nor noble agar.

As a positive control I have used HeLa cells and seeded 1000 or 1500 cells per well in a 24 well plate.
Although it only has been a few days after seeding the cells do not look viable. They look as if undergoing necrosis, ruffling cell membrane and irregular shapes.

Also when I feed the cell layer with media the cell agarose layer seem to disolve and become a geletin like substance. Is this a normal phenomenon and should resolidify after a day?

Thank you.

#2 bob1

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Posted 25 July 2013 - 12:29 PM

The reason you need to use 2x medium is that it is the correct osmolarity for your cells when diluted 1:1 with the agarose. The reason agarose is used is that it is more defined (so no extraneous stuff that could affect your cells) and typically is the low melting point one (i.e. sets at about 35 degrees rather than 45).

The above reasons are why your cells look sick.

#3 crom80

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Posted 25 July 2013 - 12:33 PM

The reason you need to use 2x medium is that it is the correct osmolarity for your cells when diluted 1:1 with the agarose. The reason agarose is used is that it is more defined (so no extraneous stuff that could affect your cells) and typically is the low melting point one (i.e. sets at about 35 degrees rather than 45).

The above reasons are why your cells look sick.


I am currently using 2x medium (hence 20% FBS) so that once I mix it with the base or cell layer it becomes 1x. Also I am using agarose for the cell layer as the protocol. The agar is used for the bottom layer.

The protocol says to warm the media and cell layer agarose at 40C. Can this be too high temp for the cells?




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