Phagocytosis of opsonized fluorescent microspheres by human neutrophils. A two-color flow cytometric method for the determination of attachment and ingestion.
A highly reproducible two-color fluorescence cytometric method is described for determining the amount of receptor-mediated and of non-specific phagocytosis by human neutrophils of polystyrene microspheres that have been covalently coated with C3b, iC3b, IgG, mixtures of these, BSA or human F(ab')2. The method includes a correction for externally bound particles and thus measures net phagocytosis. The method involves the incubation of neutrophils with coated green fluorescent microspheres in buffer alone or with cytochalasin D to inhibit phagocytosis followed by staining the externally bound microspheres with red fluorescent antibodies to the immobilized ligand, and determining the green and red fluorescence in a dual laser fluorescence activated flow cytometer. The red to green fluorescence ratio of the mixtures containing cytochalasin D allows for a correction of the green fluorescence due to externally bound microspheres in the mixtures not containing cytochalasin D. The corrected green intensities thus represent net phagocytosis. The specificity of receptor-mediated phagocytosis was confirmed by inhibition with fluid-phase C3b or iC3b or monoclonal antibodies to the receptors. The method can be applied to the determination of both adherent and suspension phagocytosis and can be used as a general model of the phagocytosis of bacteria by neutrophils.
Ogle
J D
JD
Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine, OH 45267-0522.
Noel
J G
JG
Sramkoski
R M
RM
Ogle
C K
CK
Alexander
J W
JW
D002021:Buffers; D002118:Calcium; D015638:Cytochalasin D; D003572:Cytochalasins / Q000494:pharmacology; D004306:Dose-Response Relationship, Immunologic; D005434:Flow Cytometry / Q000379:methods / Q000592:standards; D005455:Fluorescent Antibody Technique; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D008863:Microspheres; D009504:Neutrophils / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D009895:Opsonin Proteins; D010587:Phagocytosis / Q000187:drug effects; D011951:Receptors, Complement / Q000502:physiology; D017463:Receptors, Complement 3b; D012542:Scattering, Radiation; D013997:Time Factors; D000267:Tissue Adhesions
D002021:Buffers; D003572:Cytochalasins; D009895:Opsonin Proteins; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D015638:Cytochalasin D; D008274:Magnesium; D002118:Calcium