We are planning to run quite simple treated vs. untreated experiments with cells that we plan to irradiate and lyse for WB samples. Problem is that we will have a lot of samples when using different amounts of irradiation, different recovery times before lysing and different variations for the treatment, we want to have some sort of time lines for all of these parameters. Running western or cell staining for all samples is going to be a real headache. Is there any easier option available to screen large amount of samples for DNA damage?
Hi Mikko, there are quite some options. It largely depends on what you want to adress: DNA strand breaks, clastogenic or aneugenic effects, apoptosis...
The comet assay is quite a good method to detect DNA strand breaks. However, these alterations can or cannot be repaired by the cell. If you want to detect effects on the whole cell genome, you could assess micronuclei to represent clastogenic and aneugenic effects. If you are interested in apoptosis reactions, you could go for typical apoptosis markers (caspase-3 etc.) via ELISA or RT-PCR assays. Furthermore, you can use the forward-mutation to 6-thioguanine resistance (e.g. in V79 cells) which does not work in every cell-line but is by far the simplest method, comparable to the Ames assay in bacteria. As you see, there are some options. Which one you should use is largely depending on the addressed question. Best, Markus
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