Assessment of blood eosinophils activation
Whole heparinized blood was obtained from 10 ECRS patients. Blood was
incubated for 24 h at 37°C with either recombinant human CD40L protein
(rhCD40, 5 ug/ml; R&D systems, Minneapolis, MN, USA, 6420-CL-025) or
recombinant human ICOS protein
(rhICOS, 10ug/ml; R&D systems,
Minneapolis, MN, USA, 169-CS-050). IgG (5 ug/ml; R&D systems,
Minneapolis, MN, USA, 1-001-A) was used as control. Cells were harvested
for further analysis. Leukocytes were stained with an antibody cocktail
of CD45-APC (Life Technologies, Calif. USA, 17-0459-42, HI30), CD16-FITC
(BioLegend, San Diego, Calif. USA, 360716,B73.1), and CD69-PE
(BioLegend, San Diego, Calif. USA, 985202,FN50). Eosinophils were
defined as CD45+CD16- and CD69 was
determined as its activation marker.