Neurotransmitter sampling and storage for capillary electrophoresis analysis |
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Authors: | X Zhang Robert R Fuller Robin L Dahlgren Kurt Potgieter Rhanor Gillette J V Sweedler |
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Institution: | (1) Department of Chemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801, USA e-mail: jsweedler@uiuc.edu, US;(2) Department of Molecular and Integrative Physiology, University of Illinois, XX |
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Abstract: | Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the
analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before
being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral
cell and pedal ganglia homogenates isolated from Pleurobranchaea californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized
ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature
for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within
10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine
and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least
five days by combining the ascorbic acid preservation and freezing at –20 °C. The timing of preservation is critical in maintaining
the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after
isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses
less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed
within 4 h to obtain losses of less than 10% for serotonin related metabolites.
Received: 23 August 2000 / Revised: 2 November 2000 / Accepted: 7 November 2000 |
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