Page 111 - 73_02

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

P2X3 and P2X7 (2, 3). Thus, we showed that AR, in the absence of
H2O2, was quickly oxidized when BzATP was present. Furthermore,
we obtained first evidences that glutamate determinations using

Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit were also

strongly interfered by BzATP by using individual measurements in a

fluorescence emission spectra (LS55 spectrofluorimeter from Perkin

Elmer). In the present work we showed how this interference can

lead to misinterpret the results and, therefore, to hypothesize effects

that do not correlate with the ones mediated through ionotropic

nucleotide receptors (P2X) in biological systems.

TABLE 1. Examples of Amplex Red-based assays. The limits of detection of the
corresponding metabolites or enzymatic activities are shown

Metabolite Enzymatic
Activity

Amplex Red Catalase Assay Kit 50 mU/ mL
Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit 1.2 x10–5 U/mL
Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit
40 µU/mL
10 nM 0.05 mU/mL
Amplex Red Glucose/Glucose Oxidase Assay Kit 3 µM 2 mU/mL
Amplex Red Galactose/Galactose Oxidase Assay Kit 4 µM
Amplex Red Cholesterol Assay Kit 5 ng/mL 0.002 U/mL
Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit 0.2 mU/mL
0.1 mU/mL
0.3 µM
Amplex Red Uric Acid/Uricase Assay Kit 20 nM

Amplex Red Xanthine/Xanthine Oxidase Assay Kit 200 nM

METHODS

Culture of granule cells

All experiments carried out at the Universidad Complutense of
Madrid followed the guidelines of the International Council for
Laboratory Animal Science (ICLAS). Cerebellar cultures were

443