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U2OS treatment with etoposide

cell culture dna damaging agent U2OS cells

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4 replies to this topic

#1 Demi3

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Posted 16 January 2012 - 11:41 AM

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!!!

#2 bob1

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Posted 16 January 2012 - 12:02 PM

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

#3 Demi3

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Posted 16 January 2012 - 12:08 PM

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

#4 bob1

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Posted 16 January 2012 - 12:14 PM

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.

#5 Demi3

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Posted 16 January 2012 - 12:19 PM

thanks, I'll try that as well!





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