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Problems with MCF-10A adhesion and growth

Cell culture MCF-10A adhesion

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#1 zipit145

zipit145

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Posted 12 June 2012 - 11:00 AM

Our lab has been trying to start up MCF-10A populations from frozen samples, however we are not able to obtain any adhesion. We have tried remaking our media and working with uncontaminated samples that were obtained externally to no avail.

Our growth media consists of :

D-MEM (contains appropriate glutamine levels)= 250ml
F-12 Medium = 250ml
EGF (20ng/ml) = 100ul
Horse Serum (5%) = 25 ml
Gentamicin = 0.5 ml

Insulin (10ug/ml)
Hydrocortisone (0.5 ug/ml)

Many of the posts that I have read here mention cholera toxin, however not in regards to adhesion. When these cultures were previously operational my colleague working on them employed two different medium recipes, with and without cholera toxin, and said there was not a notable growth difference. The one troubleshooting point that I have come up with is our horse serum which is approximately two years old (stored at -20C), but I have little information to support this old serum being the issue.

Has anyone else encountered this?





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