UV irradiation - sensitivity to apoptosis (Jul/04/2010 )
Hi,
I'm intesresting in checking if my gene of interest (GOI) sensitises cells to apoptosis. Some pappers expose cells to 10 mJ/cm2 of UV
irradiation. I'm new to this experiment and would appreciate any advice.
How long are cells exposed to UV irradiation and after exposure are teh cells placed back in the CO2 incubator prior to apoptosis assay?
10mJ/cm2 is the same as 10,000uJ/cm2?
The best way to do this properly is to get a UV crosslinker such as a Stratalinker and put in your values (i.e. 10 mJ) and it will exposue the plate for the appropriate period of time. The length of this time will vary according to the strength of the UV bulbs, so you can not estimate it from a duration unless you know how much UV is coming out of the tubes.
Apoptosis from UV will take place within 6-12 hours, so the cells should be placed back in the incubator. You would also be advised to swap the medium for PBS before UVing and then replace the medium after the UV exposure, as the serum proteins will absorb quite a bit of UV.
bob1 on Jul 7 2010, 09:39 AM said:
Apoptosis from UV will take place within 6-12 hours, so the cells should be placed back in the incubator. You would also be advised to swap the medium for PBS before UVing and then replace the medium after the UV exposure, as the serum proteins will absorb quite a bit of UV.
Thank you very much for the useful advice. Will follow it up soon.
bob1 on Jul 7 2010, 09:39 AM said:
Apoptosis from UV will take place within 6-12 hours, so the cells should be placed back in the incubator. You would also be advised to swap the medium for PBS before UVing and then replace the medium after the UV exposure, as the serum proteins will absorb quite a bit of UV.
Hi,
I;m new to this and would appreciate your advice. How does UV cross-link help in studying sensitivity to apoptosis. Doe steh UV irradiation rsult in DNA cross-link and this cross-link of DNA induces apoptosis. what is teh association between cross-link and apoptosis?
UV results in strand breaks and C-T transversions, some level of which the cells can repain by themselves. However if the cells have too much DNA damage, then the cells will undergo apoptosis - presuming that the p53 pathway is active.
The crosslinker is just a way of adding a defined amount of UV, not actually crosslinking the DNA - which it would only really do if the DNA was extracted from the cells and on a suitable substrate (e.g. nylon membrane).