A rapid and simple photometric assay for quantification of eosinophil chemotaxis.
Various methods have been devised to assay eosinophil chemotaxis. In this review, we introduced a rapid and sensitive eosinophil chemotactic assay system based on eosinophil peroxidase (EPO) determination. Eosinophils were semipurified by dextran sedimentation followed by Percoll gradient centrifugation. Eosinophil migration was measured by the Boyden blind-well chamber technique using a 96-well multiwell chamber and polycarbonate membrane filter. The number of migrated eosinophils was assessed by determination of the EPO activity of drop-off cells, and photometric measurement was performed with a microtiter plate reader. The contribution of neutrophil peroxidase to the assay is negligible, and the assay is sensitive enough to detect 200 eosinophils. The method is both less tedious and less laborious than conventional manual methods, and it allows a large number of samples to be analyzed within 4 h. When eosinophils were exposed to 15 chemokines and their migration was assayed using this method, we found that only CC chemokine receptor 3 ligands, such as eotaxin, induced significant eosinophil migration.
Nagase
H
H
Department of Respiratory Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan.
Yamaguchi
M
M
Jibiki
S
S
Yamada
H
H
Ohta
K
K
Kawasaki
H
H
Yoshie
O
O
Yamamoto
K
K
Morita
Y
Y
Hirai
K
K
D002634:Chemotaxis, Leukocyte; D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000502:physiology*; D006801:Humans; D010544:Peroxidases / Q000378:metabolism; D010783:Photometry; D054397:Receptors, CCR3; D019707:Receptors, Chemokine / Q000378:metabolism
C515350:CCR3 protein, human; D054397:Receptors, CCR3; D019707:Receptors, Chemokine; D047209:Eosinophil Peroxidase; D010544:Peroxidases