2 replies to this topic

[dan_]

Hi everyone,

I would like to know if somebody has ever quantified oxidative stress in culture cells. If yes, where I can find a protocol/litterature?

Thanks!

[warsel]

dan_, on Jun 22 2009, 09:12 AM, said:

Hi everyone,

I would like to know if somebody has ever quantified oxidative stress in culture cells. If yes, where I can find a protocol/litterature?

Thanks!

There are several ways, fluorimetric measurement of DCF-diacetate (fluorescent dichlorofluorescin) is what I used before.
It's a pretty straight forward assay, I don't have a protocol handy but I'm sure you'll find something online.

[Bill Nye]

warsel, on Jun 23 2009, 11:40 AM, said:

There are several ways, fluorimetric measurement of DCF-diacetate (fluorescent dichlorofluorescin) is what I used before.
It's a pretty straight forward assay, I don't have a protocol handy but I'm sure you'll find something online.

I've used DCFDA before, try this on your cell line:

1) aspirate off media
2) incubate cells with 10uM dye in PBS @ 37c for 10mins
3) 2X wash with PBS
4) replenish media

- from my experience, DCF is leaky so try to work within 1hr after loading the dye
- my dye-stock is made in DMSO, I put some in PBS when I use the dye
- when loading the dye, use something without phenol red (ie PBS)

hope this helps

Edited by Bill Nye, 23 June 2009 - 04:53 PM.