I'm running a mammosphere assay but am worried about aggregation of cells, I have read that you can add methylcellulose to the media to make it semi viscous but I'm worried that will defeat the purpose of selecting for cells capable fo anchorage independent growth. Any ideas?
Aggregation is the first step in sphere formation. Methylcellulose use should be similar to soft-agar transformation assay.
I would have to disagree with Goodwin.
Aggregation is completely the opposite of what we want from this assay. If you consider that we are looking for colony forming cells (i.e. spheres are clonal entities) then any aggregation of cells will lead to a misinterpretation of results. Consider the original neurosphere nature protocol (Singet et al 2006), along with the attached mammosphere protocol you can see that aggregation of cells can occur but needs to be minimised.
Article A Detailed Mammosphere Assay Protocol for the Quantification...
Here is the paper which describes the use of methylcellulose to reduce aggregation.
Data Mammosphere Formation in Breast Carcinoma Cell Lines Depends...
Hi John, you are absolutely right, aggregation will make beautiful spheres, but will not impose the growth restriction needed to use this assay for stem cell enrichment.
For this purpose you need clonal spheroids derived from resilient stem cells.
By the way, I am the senior author in the article you commented. I suggest you take a look at our blog for further discussion http://stemtektherapeutics.com/blog/ where we specifically comment on this and other issues related to cancer stem cells.
Hi Angel,
We have been trying your method and we still see structures where we are never sure whether they have come from single cells or could still come from aggregates. Do you maybe have a 'Methods in Mol Biol'-type SOP for this?
Hi Torsten,
the key is cell density. When there are too many cells (and this depends on the cell line) you certainly will see spheres that are contributed by several cell populations. We did this by mixing cell populations labelled with GFP or DsRed; at low densities most spheres were monocolor, but at higher densities we saw increasing numbers of dual color spheres.
Best
We used MCF7 cells at 1000 cells/well in a 24-well low-adhesion plate and cultivated in 0,5ml of serum free DMEM with B27 etc. and methylcellulose. I guess this was to many as I believe you use this cell number in 6-well plates?
Certainly, it is too high. For MCF7 we use 200-300 cells in a 12 well plate.
Hi Angel, I read your paper and am trying to use methylcellulose, too. May I have the catalog number of methylcellulose? And one more question is have you ever tried to do mammosphere assay in sum159 cells? If yes, is there any special notice you recommend? Because the results I got were always dead or aggregated cells. Thank you very much!
- Badges
- Science topic