Detection of CD 14 on migrated monocytes by specific antibody: a possible quantification for blood monocyte chemotaxis.
In the present study we investigated the possibility to use antigen-antibody recognition for detection of monocyte chemotaxis in the 48-well microchamber assay. The described method is based on recognition of cell-specific antigenic determinants present on the migrated monocytes. After conventional 48-well chemotaxis, the migrated cells were incubated with an antibody against the monocyte surface marker CD14 (3C10 hybridoma). Subsequent incubation with enzyme-coupled antibodies and their substrate allowed the antigen and hence the migrated cells carrying this antigen, to be detected and measured in a microplate reader. Our results show that chemotaxis of normal blood monocytes towards the monocyte chemoattractants FMLP and MCP-1 could be detected with the anti-CD14 antibody 3C10 in combination with a horse-radish peroxidase coupled antibody, and that the optical density is a measure for cell number per well (positive correlation, r = 0.95). Incubation of monocytes with the applied chemoattractants FMLP and MCP-1 did not change the CD14 expression as was determined by FACScan analysis. Therefore we conclude that it is possible to use antibodies directed against antigenic determinants like CD14 to detect blood monocyte migration in a more objective way compared to subjective counting of cells on a filter. Eventually, this method can be valuable, especially for chemokine research since chemokines exert their effects on specific target cell populations. By varying the detection antibody, other cell populations besides monocytes may be quantified.
Tekstra
J
J
Vrije Universiteit, Faculty of Medicine, Dept. Cell Biology and Immunology, Amsterdam, The Netherlands.
Tuk
C W
CW
Beelen
R H
RH
D000911:Antibodies, Monoclonal / Q000737:chemistry; D000918:Antibody Specificity; D002452:Cell Count; D002469:Cell Separation; D018932:Chemokine CCL2 / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology*; D009000:Monocytes / Q000737:chemistry* / Q000276:immunology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D018932:Chemokine CCL2; D018950:Lipopolysaccharide Receptors; D009240:N-Formylmethionine Leucyl-Phenylalanine