Hi all
I am trying to make ht29 resistant cells with gradually increasing concentration of 5fu.The problem is that the cells don't grow, its about 2 weeks that confluency of cells is 40% , they don't die in 10uM 5fu but they don't proliferate as well. Dose anyone know what should I do to increase my cells.
Hi Mahshid,
It is very hard to grow colon cancer cells resistant to 5-FU. In our lab we have generated HCT-116/5-FU resistant but that took around 9 months.
Try not to always keep 5-FU treatments on the cells, put drug-free media and monitor growth of cells. and never split in 5-FU-containing media.
Hope this works,
Patrick
hi
you can detach your cells from T25 or T75 flask and transfer to 12 or 6 well plate with minimal volume culture medium for growth factor concentrated & increase cell to cell signaling . also you can used Sodium pyruvate as 1mM final concentration in culture medium for increase cell growth .
as parallel you can cell cloning "HT29" in 96 well plate for getting single cell and then test minimal to maximal ratio of 5-FU in culture medium , finally you have several clones with different tolerance to 5-FU .
good luck
Hi Mahshid,
We have published an open access article about trying to establish drug resistant models of cancer. I include the link the article here. There are several factors that need to be taken into consideration when trying to make resistant models not just drug concentration. Hopefully the article will give you a few ideas how to proceed?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944788/pdf/fonc-04-00040.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944788/pdf/fonc-04-00040.pdf
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