Hi,
We are trying to do soft agar assay in six well plate and performing on MCF7 breasr cancer cells. We are preparing bottom layer with 1.5 ml 0,8% agarose land top layer with 0.35% agarose. At the end the same/next day tried both way we pour 1ml media to feed the cell.
The problem is when we pour media on the top, agarose layer detaches and media seeps down to the bottom.
Any suggestion would be highly appreciated.
Abdul,
You can add 250 ul medium on top of each well of soft agar plate (for 6-well format), once a week to maintain moisture-will definitely work!
May i Know for what purpose you are doing soft agar colony formation assay? with MCF 7 cell line you can also go with normal colony formation assay.
Depending on the cell-line maybe you could try to increase the agarose-concentration of the top-layer a bit? But in principle the protocol you describe looks ok to me. A difference is that I don't wait too long before adding the media to the dishes (I let the dishes sit for approx. 30 minutes @ room temperature before adding the media).
Maybe you should try some other agarose-brands for this assay?
Good luck, Christian
I use 1% agarose for the bottom layer and 0.5% for the top. Similar to Christian, I only wait 20-30 minutes before I gently add growth media. Are you having an issue with the top and bottom layers separating from each other or both the layers coming loose from the plate?
thanks christian and seth!
My issu is that media get underneath the agarose bottom layer.
My another question is compulsory to add media on top? Can we grow cells without adding media on top or we add very less amount may be 400ul per well just to maintain moisture?
Abdul,
You can add 250 ul medium on top of each well of soft agar plate (for 6-well format), once a week to maintain moisture-will definitely work!
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