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untreated microplates. The modified
surface prevented the adherence and
consequent activation of PMNs. Neu-
trophils in the two types of plastic
microplates were shown to have a sta-
tistically significant difference in basal
as well as stimulated levels of superox-
ide production. Absorption measure-
ments were made using dual
wavelength measurement at 550 nm
(1 nm bandwidth), the absorption
maximum of reduced cytochrome c,
and 557 nm (1 nm bandwidth), an
isosbestic point. The authors observed
that the difference values (550
557 nm) significantly increased repro-
ducibility and sensitivity (detection
limit) as compared to the single wave-
length measurement at 550 nm.
Kinetic microplate assay for superoxide
production by neutrophils and other
phagocytic cells
Methods in Enzymology 196: 567575 (1990).
Laura A. Mayo and John T. Curnutte.
Department of Molecular and Experimental Medi-
cine, Research Institute of Scripps Clinic, La Jolla, CA
92037 USA.
Summary. This review describes meth-
ods of isolation and use of human neu-
trophils in assays for superoxide
production. The kinetic microplate
assay for superoxide follows the reduc-
tion of cytochrome c at 37 °C in a
Molecular Devices microplate reader
fitted with a special narrow (1 nm)
bandwidth 550 nm interference filter.
Single well illumination and use of
AUTOmix in whole cell assay are
reported to be valuable. Analysis of
nonlinear kinetic data with SoftMax
Pro software is presented.
Summary :
Absorption measure- ments were made using dual wavelength measurement at 550 nm (1 nm bandwidth), the absorption maximum of reduced cytochrome c, and 557 nm (1 nm bandwidth), an isosbestic point. The kinetic microplate assay for superoxide follows the reduc- tion of cytochrome c at 37 °C in a Molecular Devices microplate reader fitted with a special narrow (1 nm) bandwidth 550 nm interference filter.
Tags :
bandwidth,microplate,superoxide,assay,kinetic,production,550,well,cytochrome,difference,molecular,absorption,neu