Thanks to all who've written. Here're my replies to your queries...
>We have fairly extensive experience in recording from acutely dispersed
>sensory neurons (1.e. 1-2 days after dispersion), and have used almost
>exactly the same solutions as yourself (and there is also tons of literature
>using these solutions as well) so I doubt that is the problem. Bubbling with
>O2 should not be necessary. Some people warm the extracellular solution in
>the incubator before transfer into this solution as sometimes the neurons
>will lift off the coverslip when suddenly plunged into cold room temp
>solution.
Right. I have tried warming the Tyrode's to 35ºC,
to no (positive) effect. And the cells are not
falling off the coverslip.
By dead I mean blebby, ragged appearance with
visible nuclei, leaky and very depolarized.
>When you say the cells are dead what do you mean? Are the cells lysed? Do
>they lift off the coverslip? Or do they just not have good healthy resting
>membrane potentials? What is the evidence that they are vaible to begin
>with?
The cells look great initially- after I bring
them from the cell culture hood to the recording
chamber, they have smooth membranes, with small
dimples in them signifying the nucleus. Then,
almost before my eyes, they begin to look worse
and worse.
It is possible that they're sick to begin with,
as the initial sign they're bad is the resting
mV. Then, morphology begins to follow physiology
and the cells display the signs I mention above.
A colleague, however, is obtaining useful
morphological data from these cells, so I know
they're healthy shortly before she fixes them for
analysis, and that's roughly the same age as the
ones I use for physiology.
>>>So they all die, as soon as you take them out of the incubator? Or do
>they die only after you patch them. If it's the later, I suspect your
>electrode, pipette solution, or break in technique. If it is the
>former, then I have no idea. My only suggestion is to half, or quater
>your calcium chloride, and drop your KCl down to 2.5mM. Oh, and try
>30mM glucose.
They're dying within 15 - 20 minutes after
removal, and its en masse-- all of the cells
begin to look dreadful, so its definitely not the
internal solution.
>I know they sound like silly changes, but anything is possible?
>>(my cell culture extracellular saline (mM) NaCl 142, KCl 2.5, CaCl,
>2-0.5, MgCl 2, HEPES 5, Glucose 30. pH 7.4)
>
Thanks again.
Brad.
--
Department of Neurobiology & Physiology
2205 Tech Drive, Hogan 2-160
Northwestern University
Evanston, IL 60208
http://homepage.mac.com/bradleymcooke/index.html
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