A flow cytometric method for the detection of intracellular basic proteins in unseparated peripheral blood and bone marrow eosinophils.
Eosinophils and their basic proteins play a major role in allergic disease and methods are required to monitor their expression in clinical situations. In this article we describe a flow cytometric method for the detection of intracellular eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in unseparated clinical samples. After fixation with parabenzoquinone and permeabilization with n-octyl-beta-D-glucopyranoside, the detection of intracellularly stored proteins was achieved using of monoclonal antibodies against ECP (EG1, EG2) and EPO in combination with an FITC-labeled second step antibody. Confocal microscopy was used to demonstrate the intracellular origin of the fluorescent signal. Fixation with parabenzoquinone was superior to a previously described protocol using paraformaldehyde, since it reduces non-specific binding of FITC to the basic proteins in eosinophils. Fixation and permeabilization do not alter the light scatter characteristics of eosinophils in contrast to other leukocytes and thus permit gating on eosinophils without prior purification. Furthermore, the procedure does not alter the detection of cell surface antigens on eosinophils and simultaneous measurements of surface antigens and intracellular proteins is possible. We have used different clinical samples (peripheral blood, bone marrow cells) to demonstrate differences in the expression of ECP and EPO. We conclude that the detection of intracellular eosinophil proteins by flow cytometry is a rapid, easy and semiquantitative procedure which may be used to study their expression in diseases where eosinophils are involved.
Krug
N
N
Department of Paediatrics, Ruhr University, Bochum, Germany.
Thurau
A M
AM
Lackie
P
P
Baier
J
J
Schultze-Werninghaus
G
G
Rieger
C H
CH
Schauer
U
U
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis; D016227:Benzoquinones; D001798:Blood Proteins / Q000032:analysis* / Q000276:immunology; D001853:Bone Marrow / Q000737:chemistry / Q000201:enzymology; D001854:Bone Marrow Cells; D047091:Eosinophil Granule Proteins; D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000737:chemistry* / Q000201:enzymology*; D005069:Evaluation Studies as Topic; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005960:Glucosides; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry / Q000201:enzymology; D018613:Microscopy, Confocal; D010539:Permeability; D010544:Peroxidases / Q000032:analysis* / Q000097:blood / Q000276:immunology; D012260:Ribonucleases; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D016227:Benzoquinones; D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D005404:Fixatives; D005960:Glucosides; C018619:octyl-beta-D-glucoside; C004532:quinone; D047209:Eosinophil Peroxidase; D010544:Peroxidases; D012260:Ribonucleases; D016650:Fluorescein-5-isothiocyanate