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Sorry for the long post...
I'm working with several species of plant-parasitic nematodes and would like to
measure their genomic DNA content by flow cytometry. As a first test, I thought
I could run C.elegans, as it is well-characterized and would serve as an
appropriate control. However, I've had a bit of difficulty (=>understatement)
getting clean readings from C. elegans (haven't even *tried* my worms yet -
they are difficult to obtain in large numbers...). I'm working with the
cytometry facility here on campus (NCSU), so I'm not doing the flow work myself
and have little understanding (yet!) of the technical aspects of data
collection. I just know that my samples are not giving "clean" readings; there
is lots of flourescent "junk" making interpretation imposssible.
My problem seems to be with the preparation methods. Normal preps (as for
animal tissue) do not seem to disrupt the worm cuticle (after detergent,
trypsin and RNase I get wriggling, intact worms). Then I thought maybe I should
just make a nuclear prep, so I used a method from people who get nuclei for
transcription(?) experiments. This included grinding the *frozen* worms and
several pelletting steps to isolate the nuclei from cellular debris. No good -
I *thought* the nuclei looked OK under the 'scope, but still too much
variability (however, these nuclei were in the 'fridge for a few weeks before
the cytometry, and were *not* fixed). Now I'm trying to grind the worms in
sand, and layer the extract on Percoll to get nice clean nuclei, but under the
'scope it looks as if I have lots of junk.
I'm not sure if I'm even going in the right direction here. The cuticle seems
to be my main problem - if I don't "bash" the worms pretty hard, I can't
liberate nuclei, but if I get the worms opened, I seem to get lots of
flourescent trash. The worms are about 1mm long (at the biggest); their cuticle
is made (mostly) of collegen. I considered using collegenase treatment but I've
been told that the outside of the worm's cuticle is resistant to it (with *big*
nemas, people have injected collegenase into the worms to digest them). I still
may try soaking them in collegenase, but I was hoping someone would have some
ideas or (better!) have worked with nemas before. I've looked though the
literature, and don't find anything on looking at worm cells by flow cytometry
(although individual *eggs* have been counted).
Any suggestions? Should I carefully redo the nuclear prep and fix the nuclei
before staining them? Try collegenase? Maybe I could heat the worms in the
microwave and "pop" them... (sounds gruesome, but I'd try anything now...).
Thanks lots for any help or ideas you can give. Please respond via email as I
don't get mail from this list. Thanks!
-- Susan http://www4.ncsu.edu/unity/users/s/sjhogart/public/home.htmlHer face hangs in portrait on the post-office wall; She's stuck in my heart now where my blood belongs. TMBG
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