Very Low Counts with 3H Thymidine Assay / Millipore Multiscreen system

David Liu via methods%40net.bio.net (by amanosz from yahoo.com)
Wed Nov 14 21:12:58 EST 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


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