cholera toxin neccessary for culturing MCF10A? - (May/18/2009 )

I've been working with two lines of MCF10A and have been haven't been able to pass them. Trypsinization seems to be very difficult relative to MCF7 or MDA-231. Once plated, they do not adhere very well and do not spread. I have tried two types of media.

A proprietary media from Lonza/clonetics MEBM + MEGM 'bullet kit' (recommended by ATCC), though I did not include the recommended cholera toxin.

I also made DMEM/F-12, 5% horse serum, insulin, pen-strep and hydrocortisone. Again, this is missing the recommended cholera toxin.

Is the lack of cholera toxin, what is causing the problem. I've ordered it, but haven't received it yet, though I suspect it would enhance the growth of these cells and did not think that it would be required for their spreading.

I would really appreciate any advice from someone who may have experienced something similar.

Thanks!

-boscell-

Hi, Did you add EGF?

as MCF-10A is not a cancerous cell line it's dependent on external EGF. if you don't add it won't grow.

-hafezparast-

in case of trypsinization and other things read this

https://brugge.med.harvard.edu/Protocols/Ca...atrigel.doc.pdf

maysam

-hafezparast-