Can you provide a protocol for treating cells with UV?

We perform our UV treatments as follows:
 

1. Adherent cells are cultured until they reach a confluence of approximately 80%.
2. For the UV treatment:
   1. We remove the growth media from the plates and set it aside in a sterile container.
   2. Then, we treat the cells with UV using a crosslinker (e.g., UVP CX-2000 UV crosslinker). The covers are removed from the cell culture plates when placed in the crosslinker. The cells are treated with UV as recommended (e.g.., 100 mJ/cm2), regardless of the size of the culture dish (the UV amount is typed into the crosslinker, and the treatment/timing is done automatically based on the value inputted into the machine).
3. Immediately after treatment, the media is replaced on the cells and the cells are allowed to recover in the incubator for the recommended time (e.g. 1-hour) before lysing.
4. The cells are washed with ice-cold 1X PBS and lysed with your buffer of choice (we usually use 1X #7722 blue loading buffer SDS+DTT, but you may prefer to use 1X #9803 CLB + PMSF or other lysis buffers). We typically lyse a 15cm plate in 0.5mL - 1mL lysis buffer.
5. The lysate is removed from the plates and sonicated on ice using a probe tip sonicator (3 times for 15 seconds each).
6. If the samples are prepared in SDS +DTT lysis buffer, the lysate is boiled at 95 - 100C for 5 minutes before use.

Last updated: September 12, 2024