I have been trying to standardize the protocol using 1% agarose for the mammosphere assay. I'm observing that even at very low seeding density the cells tend to aggregate or clump together. It would be of great help if someone could share a protocol for teh mammosphere assay using agarose
You can seed 500 to 1000 cells per well in serum free media and adding a cocktail of growth factors like EGF (10ng/ml), FGF (10ng/ml), Insulin (20ng/ml), and Lif1 (10ng/ml).
Be sure to change the media every 2 days. Spheroids will start forming in 4-5 days and can then be counted under a light microscope. Certain spheroids may start clumping together, to avoid this be sure to agitate your plate properly so as to be sure that the cells are well dispersed in the medium.
Hope this helps.
Thanks Amirali. I have been using serum free media with the supplements as suggested. Any suggestions on the optimum percentage of agarose to be used?
I have personally always used 1% agarose. However, you could also try standardizing with 1.5% or 2% agarose at the max.
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