Glutamate release

Michael Duggan michael.duggan at camr.org.uk
Wed Jan 22 05:11:06 EST 1997


Dear All,

We want to measure glutamate release from cultured dorsal root 
ganglion neurons. We have had some tries with radiolabelled D-Asp 
and glutamine labelling of the cells, but the results are not as good 
as we would like them to be (variability in loading, lack of calcium 
dependence for release etc.).

We would, therefore, like to try measuring the release of  endogenous 
glutamate. The question is which is the best way to measure this? We 
have tried using glutamate dehydrogenase and NADPH fluorescence, but 
the signal was at the limit of detection. Obviously we could increase 
the signal by increasing the number/density of the cells but we are 
going blind doing the dissections already!

There are methods in the literature using HPLC of derivatised 
(o-phthalaldehyde, for example) samples but before we invest in enormously
 expensive fluorescent detectors, can anybody answer the following 
questions:

1.    Are the hplc methods more sensitive than the enzymatic methods? 
       If so by how much?

2.    What are the best derivatisation reagents for this technique?

3.    Are there any hplc methods which would allow sensitive detection by 
       absorbance rather than fluorescence (we have a UV detector 
       already, so this would be a lot cheaper)?

4.    Does anybody have any other good ideas about how best to do 
       this?

Thanks for any help you can give us

Mike and John

Michael J. Duggan,
Centre for Applied Microbiology and Research,
Porton Down,
Salisbury,
Wiltshire,
SP4 0JG,
UK

michael.duggan at camr.org.uk

Tel 01980 612733
Fax 01981 612100



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