[Neuroscience] Re: Pertussis toxin?

jonesmat via neur-sci%40net.bio.net (by jonesmat from physiology.wisc.edu)
Tue Jul 31 00:33:56 EST 2007


On Jul 27, 5:19 am, Bill <connelly.b... from gmail.com> wrote:
> I'm starting to feel really guilty for asking so many questions on
> this group, but the answers are often so good I can't resist.
>
> I'm investigating a novel G-protein coupled receptor in cortical brain
> slices and I want to see whether it works via Gi, Gs or Gq. I remember
> simple pharmacology, If pertussis toxin blocks the response of the
> agonist, it's Gi mediated. The methods I found are a bit
> heterogeneous, anywhere from 2 to 20 hours of incubation with 2-5ug/mL
> of pertussis toxin at 22 to 37 degrees.
>
> That was fine, I could empirically figure out how long I needed to
> incubate the slices by using a well established ligand (I was thinking
> baclofen), -BUT-
>
> My holding chamber holds about 70mL, to fill that with 2ug/mL
> pertussis, that would require 140ug of pertussis. 50ug of pertussis
> toxin cost $500 US from sigma (only $170 from List Biological Labs);
> so that's $1,500 a day (or $500).
>
> Either way, that is big big money in my books. Can anyone either a)
> see a mistake in my logic or b) figure a way around this problem?



I reckon the real problem is how to keep a very small volume (e.g., 2
ml) properly oxygenated for several hours, so that slices don't become
ischemic while you incubate them in PTX?

How about using a small 1-2 ml airtight sealable vial? Fill all the
way to the lid with PRE-oxygenated slosh, containing PTX, plunk in the
slice, then screw on the airtight cap. No deadspace, no loss of
oxygen.

Could check this method easily to see if slices in control ACSF are
still viable after a couple hours of this storage. Worth a try at
least, eh?

On the other hand, I've had a long standing suspicion that sitting in
even a large oxygenated holding chamber for several hours is WORSE
than being on the rig under continous perfusion. The evidence being
that the first slice you put on the rig always seems to be the
healthiest one. I.e., even after a few hours, when you go to get a new
slice fresh from the holding chamber, it never looks as good as the
one you JUST removed from the rig! My hypothesis (i.e.,
unsubstantiated fantasy) is that the slices are constantly releasing
"bad stuff" (they are in the process of slowly dying, after all), and
sitting around in this same "bad stuff" leads to "bad things". Whereas
the first slice on the rig has had its "bad stuff" continuously
removed by the rig perfusion.

Do you have any experience with perfusing the holding chamber itself?

BTW, if any of this works, definitely let me know!

Matt


PS - Sorry for responding so consistently to your highly varied
questions. I'm actually not THAT much of a know-it-all. But yours are
among the only questions in this newsgroup anymore that I even
understand, let alone can contribute to.












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