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VOL. 73 (2), 441-452, 2007 GLUTAMATE DETERMINATIONS USING AMPLEX RED...

fluorescence showed the following values, showed in Figure 4: Glu
0.1 µM, 909 ± 128 a.u. (n = 3); Glu 0.5 µM, 2713 ± 347 a.u. (n = 3);
Glu 1 µM, 5621 ± 257 a.u. (n = 3). All these values were significantly
enhanced in presence of 10 µM BzATP (Glu 0.1 µM, 1851 ± 304 a.u.,
n = 3, p < 0.01; Glu 0.5 µM 4362 ± 294 a.u., n = 3, p < 0.01; Glu 1
µM, 806551 ± 407 a.u., n = 3, p < 0.01) and 100 µM BzATP (Glu 0.1
µM, 5904 ± 935 a.u., p < 0.001; Glu 0.5 µM 8216 ± 1103 a.u., p <
0.001; Glu 1 µM 12371 ± 461 a.u., p < 0.001).

FIGURE 3. (a) BzATP interferes with Amplex® Red Glutamic Acid/Glutamic
Oxidase Assay Kit. Granule neurons (14 days old) were stimulated for 1 min with
several concentrations of BzATP (100 nM-200 µM), in absence of magnesium, and

the medium was taken and the glutamate content was measured using Amplex®
Red Glutamic Acid/Glutamic Oxidase Assay Kit. As it can be observed in this
graph, BzATP induced an increase in resorufin fluorescence that was dose-

dependent. (b) Graph show that neither BzATP nor ATP stimulation induce pore
formation in granule neurons in culture. As positive control, granule neurons were

permeabilized with triton and an increase in YOPRO fluorescence could be
detected.

Data reported in this work confirm that BzATP interferes the
measurement of glutamate using Amplex® Red Glutamic Acid/
Glutamic Oxidase Assay Kit. Thus, the measurement of glutamate in
the media after stimulation with BzATP seems to indicate that P2X
induced pore formation through which glutamate reached
extracellular medium. However, the resorufin fluorescence was not
due to an increase in glutamate content but BzATP interfered on
Amplex Red oxidation catalyzed by HRP. These data are really

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