Does anyone irradiate their cells? - (Mar/31/2014 )
Every week I gamma irradiate both L6 myoblasts and vero cells. A lab that I did some training at exposes their cells to 2000 rads in order to stop them from growing. I've been hitting my cultures with 2000 rads in the irradiator we have here, but for some reason they keep dividing, grow beyond a monolayer, then start detatching.
This past week I did not infect one of the cultures that I irradiated so that I could monitor how many generations they kept growing for, only to get in today to find out that all the cells are detatching. So much for that idea. It seems that Trypanosomes do something to the cells that encourages host cells to stay adherent (they also seem to make irradiated cells live longer).
Does anyone else here irradiate their cells? What kind of cells do you use? What's your irradiation dose? What's the source? Do you irradiate with the cells attached to the flask or do you detach them first?
I'm curious to see what other people do, as there are very few people here that I can compare protocols with.
It's not comparable to gamma, but I have used 20 W/cm2 (IIRC, it might have been /m2) for UV light in the past on a range of different cancer cell types.
Check the p53 status of your cells, if p53 isn't there or is inactivated then the cells will struggle to enter apoptosis upon DNA damage.
What would be the best way to check for p53? I'm guessing I'd need to do a Western.
edit: Actually, I don't want the cells to enter apoptosis. I want them to be alive so that I can infect them. I'm irradiating them with the intent of damaging their DNA to the point that they are unable to replicate, but are also able to stay alive until the parasites rupture them.
p53 westerns would work - you should see it up regulated within 8 hours after irradiation.
Check out methods of making feeder layers if you need protocols for this sort of thing.