5-Aza treatment - (May/18/2005 )
HI,
I am new to 5-Aza treatment of cultured cells.
I have treated my cells with different concentration of 5-Aza.
Even at concentration as high as 20 microM, the growth
of cells does not seem to be greatly affected.
Could anybody tell what is the simplest way to determine if 5-Aza
is working.
I dissolve 5-Aza in PBS at 100mM and keep it in 100 microL
aliquats at -70C.
Thanks.
hi slab,
how long do you treat your cells for?
I have titrate the drug over at lease three days. The range is usally 0-20uM. Each day I replemish the drug as it has a short half life.
I then do a viability assay and you should see a marked drop in viability of your cells at the end of your timepoint.
However you won't see this if your cells are resistant (ie: they are actively expressing and producing cytidine deaminase).
good luck!
Nick
Well, unfortunately, my cells seem a bit resistant to usual concentrations of 5-Aza treatment.
On MSP, treatment of cells with 20uM 5-Aza for 3 days suggests a weak demethylation, though.
Should I give up these cells and move on to other cells, or are there any other solutions, for example, increasing the concentration of the drug or using inhibitors of cytidine deaminase?
Thank you for your help.
you can try and go higher, but 20uM is the highest I have seen people go in the literature.
is there any indication in the literature of people treating cells with this drug?
If this doesn't work then it will be worth your while switching cells.
I had a colleague trying to treat HT1080 cells with 5azaC to no avail, he switched cells and managed to kill them all!
Nick
Thank you.
By the way, do you have any idea what types of cells tend to be resistant to 5aza treatment, e. g. epithelial cells vs. stroma cells?
Hard to tell you.
If you can assay for cytidine deaminase activity (the enzyme responsible for the resistance) that would tell you if your line is resistant or not.
Nick