4 replies to this topic

[Demi3]

Hello everyone.

I am trying to treat U2OS cells with Etoposide to induce p53 levels for quite a long time now but it seems that most of my cells die. Does anyone know a valid protocol to do that? None of the articles that I have read refer to the type of plate (dish 100cm?, 6 well plate? and the number of cells that I should start with.  Do I leave the drug until the time I harvest my culture ? I am not sure if I have to remove the drug and then incubate for 24h before trypsinisation. What is an ideal concentration of etoposide and an ideal incubation time? Do I also collect the floating cells at the end?

Thank you!!!

[bob1]

The following is the information I have on Etoposide, hope it is useful:

Etoposide (VP-16)
Topoisomerase II inhibitor. Stabilizes the covalent complexes of topoisomerase II
with DNA. Has major activity against a number of tumors, including germ cell
neoplasms, small cell lung cancer, and malignant lymphoma. Induces apoptosis in
mouse thymocytes and HL-60 cells. Activates PKCα.
Soluble in: DMSO
Stock concentration: 100 to 500 mM (store at room temperature)
Working concentration: 50 to 200 µM
Duration of incubation: 1 to 24 hr

[Demi3]

hmmm, at least now  I know that I can not leave etoposide for >24h and I should start optimisations between 50-200μM. Thank you:)

[bob1]

Further note that p53 expression is rather transient and quite quick (approx 8 hours post insult).  I have found that the best/easiest way to induce p53 is by irradiating with UV at 10 mJ/cm², and then harvesting between 6 and 10 hours post irradiation.

[Demi3]

thanks, I'll try that as well!