thymidine incorporation assay - (Nov/09/2004 )
I have been trying to do a 3[H] thymidine incorporation assay with CD4+CD25- cells isolated from mouse spleen. Unfortunately, I have been very unlucky. I plated 2.5 x 10^4 T cells and the same number of APC (T cell-depleted, 3000rads irradiated splenocytes) in 96 well plate pre-coated with 10ug/ml anti-CD3 Ab, and cultured for 72 hrs. Then I pulsed with 1uCi/well [3H] thymidine for 18 hrs, harvested on a cell harverster onto a glass filter, let dry, and read the next day. But for some reason, my reading was very low, so low that it seemed to me there's no proliferation at all (somewhere between 2 to 100). I have been very frustrated with it. One person has suggested that 10ug/mL CD3 is too concentrated, and maybe T cells are activated and underwent apoptosis, that's why I didn't see any proliferation.
Does anybody have similar experiences and would like to share some successful secrets with me?
Thank you very much!
One point I'd like to make is that if your thymidine is a bit old, this can present a problem with the sensitivity of the assay.
Although the halflife of the isotope is quite long, the thymidine itself can decay to the point where it isn't incororated into the dividing DNA with much efficiency. We have found that after 6 months of storage, decreased signals become increasingly common.
In figuring this out, observing the cells under microscope can tell you whether you have good division taking place - clustering of dividing cells everywhere would indicate you are getting division without a signal, whereas a homogenous mass of single cells in the middle of the well might suggest lack of division. I'd be surprised if you aren't getting polyclonal responses with CD3 and APC together, this usually gives a powerful response.
Hope this helps!