I need a positive control for an immunocytochemistry staining for 8-oxoguanine and gamma-H2AX (dsDNA breaks). I was thinking about UV-light irradiation. Does anybody have a protocol or advice regarding the dose, duration of irradiation and time after irradiation to let the damage occur (but not to be repaired)? I have a 8 Watt lamp with the possibility to use a wavelength of 254, 302 or 365nm.
Thanks very much,
Adrià
Irradiating samples in the UVA-UVB range should provide you with ample induction of DNA damage, including CPD, 6-4PP and 8-oxo-dG, as well as ss and ds breaks. I don't know what model type you are inducing DNA damage in (ie, cells, cell free system, etc), but using shorter time points (anything before 24h) should be good. Depending on the cell type, certain repair, particularly of 6-4PP can start as early as a couple hours post-UV and are usually gone by 24h. So you may need to do a time-course and see. As far as dosing, it really depends. Most people measure in one of two formats: J/m2 (kJ/cm2) or MED. To determine what dose you will give, you need to use an appropriate meter and this will help you figure out how much irradiation you are introducing to your samples.
What should the positive control in your experiment tell you? Are you looking for cell cycle arrest, mitosis, or apoptosis? UV light induces (indirectly) only few DSB and there are published papers for the differences in UV vs. X-ray and DSB formation (e.g. this one from my previous lab: DNA Repair (Amst). 2011 Jan 2; 10(1): 5–15.). If you absolutely need DSB, and the UV source, UV dosage, and measurable endpoint seems to be problematic, and you cannot use X-ray as alternative DSB inducer, you can treat your cells with Mytomycin C or Etoposide to induce double strand breaks. Simply add to medium (e.g. 10uM) and within 1h you'll see phosphorylated H2AX using normal immunohistochemistry protocols.
Anne and Christiane,
Many thanks for your replies, they have been really useful. Just to clarify:
-I’m using rat primary neurons.
-About the units for dosing, if I know the J/m2 I want to give and I know the W/m2 of the lamp –I do– then I can work out the time of exposure, as 1W=1J/s, right?(J/m2 can’t be equivalent to KJ/cm2 though, maybe to 100uJ/cm2?).
-The positive control is just for the staining itself: I want to optimize the use of antibodies against 8-oxoguanin and gamma-H2AX (or other robust readouts for oxidative stress) for a future use as phenotypic readouts, and in order to make sure that I can detect these markers, I wanted to treat some of the cells to induce the oxidative stress response.
-I knew about Mytomycin C, Etoposide and some other compounds that induce DSB, but I wanted to use a treatment that allowed for the detection of both markers (8-oxoguanin and gamma-H2AX). What I didn’t know is that, by treating with UV light and measuring gamma-H2AX, I wouldn’t be looking at DSB but just the H2AX phosphorylation (I thought it was biologically equivalent), so good to know! So, if I used etoposide, I still would have to use another treatment to boost the 8-oxoG. I guess that X-rays would be a better solution then, as it would induce both phenomena.
Thank you again!
Adrià
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