Concurrent measurement of antigen- and antibody-dependent oxidative burst and phagocytosis in monocytes and neutrophils.
The current study aims to review flow cytometric (FCM) parameters for the quantification of phagocytosis. A limitation of existing methods is their difficulty with accurate quantification of the phagocytic index, i.e., number of beads per phagocyte, in individual cell lines in mixed cell suspensions. We have quantified phagocytosis and the oxidative burst simultaneously using fluorescent beads coated with meningococcal outer membrane vesicles (OMV beads) by the conversion of dihydrorhodamine 123 (DHR-123) to rhodamine 123 (R-123). Both these processes depend on specific serum opsonins. After the incubation, staining with a fluorescent anti-CD14 monoclonal antibody succeeded in discriminating phagocytosing monocytes from neutrophils. The spectral overlaps between OMV beads, R-123, and anti-CD14 could be completely compensated. Percentage of phagocytosis and the phagocytic index were similar in monocytes and neutrophils, but the oxidative burst behaved differently. Two monocyte subpopulations were observed. Both subpopulations spontaneously converted some DHR-123 into R-123, whereas the reaction was triggered by phagocytosis in neutrophils. The total oxidative response increased with increasing phagocytic index in both cell types, but the oxidative burst in monocytes was about twice that of neutrophils. The oxidative ratio (mean R-123 fluorescence value divided by the phagocytic index) declined with time in monocytes, but increased in neutrophils. Our results demonstrate the need for careful attention to technical details. This single-laser, three-color FCM method facilitates the comparative research of phagocytosis and the oxidative burst in monocytes and neutrophils and provides a basis for a number of applications in hematology, infectious medicine, and immunology.
Bassøe
C F
CF
Division of Hematology, Haukeland University Hospital, Bergen, N-5021, Norway. cfba@haukeland.no
Smith
I
I
Sørnes
S
S
Halstensen
A
A
Lehmann
A K
AK
D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology; D002448:Cell Adhesion; D002478:Cells, Cultured; D018920:Coculture Techniques; D003116:Color; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000032:analysis / Q000276:immunology; D008297:Male; D008863:Microspheres; D008875:Middle Aged; D009000:Monocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D009345:Neisseria meningitidis / Q000276:immunology; D009504:Neutrophils / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D009895:Opsonin Proteins / Q000276:immunology; D010587:Phagocytosis; D016897:Respiratory Burst; D012235:Rhodamines / Q000378:metabolism; D013997:Time Factors
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D001425:Bacterial Outer Membrane Proteins; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D009895:Opsonin Proteins; D012235:Rhodamines; C058319:dihydrorhodamine 123; D017493:Leukocyte Common Antigens