Low cpm for 3H Thymidine Assay / Multiscreen - Something wrong with my wash and ppt steps? (Nov/14/2007 )

Hello,

Does anyone have experience using the Millipore Multiscreen system for 3H-Thymidine incorporation assay? I am having trouble with getting very low cpm's for my assays. I get at most ~5000 cpm under proliferating conditions which is higher than no-growth controls (~2700), but nowhere near the published 20,000-50,000 cpm that I am expecting to see. I think I've narrowed it down to the cell harvesting, wash and precipitation steps, and could use any help, ideas, or suggestions.

I'm using a T cell line, Kit225, which is IL-2 dependent for growth. I know they are proliferating and that my IL-2 batch is good, because I use the same conditions to maintain growth. Also, cell density is visibly higher (using microscope) under proliferating conditions at the end of 48 hours. I am following published protocols for growth and pulse times (48 hours growth, pulsing with 1uCi 3H for the final 16-18 hours). This is actually on the high end for growth and pulse times, compared to published protocols, so I should be capturing S phase and I should see 3H thymidine incorporation.

I am following an old lab protocol that was used for primary T cells for the harvesting and wash steps. I am using the Multiscreen system from Millipore, with a vacuum manifold and a punching apparatus, and am using Millipore Durapore HV 0.45um filter 96 well plates.

My protocol is as follows:
1) Prewet membrane, vacuum, load cells
2) Wash 2x with PBS
3) Add 5% TCA, and precipitate 20 min on ice, then vacuum filter out
4) Dry membranes, and punch into vials with 0.5mL bleach diluted 1/12 times.
5) Agitate on rotary shaker 30 minutes, then add scint fluid (Ultima gold XR) and count.

I have also tried precipitating with 20% TCA, and 100% Ethanol. All wash and ppt solutions are ice cold. This is also very similar to the protocol that the Multiscreen literature recommends, though they use a different cell line, and they add ethanol washes after precipitation.

As I mentioned, I'm quite confident the cells are proliferating, so I don't know where I could be going wrong. The only possibility is in step 5, which according to Millipore literature, bleach is required to release incorporated 3H from the Durapore membranes. However, the rotary shaker is not vigorously agitating (not like a tabletop vortex), but rather the speed of a shaker you would use to wash protein gels or western blots. However, I don't see how this can be causing my counts to be 10% of expected values.

Does anyone have any ideas or suggestions on what I should try next or how to troubleshoot this?

Thank you very much in advance!

David

Just wondering...

Do your department/institute have a TopCount?

If yes, then you do not need to do step 2-4... only need to add the scint fuild to the membrane... put the membrane in the plate and read.


Hope this may help.