Adhesion of subsets of human blood mononuclear cells to endothelial cells in vitro, as quantified by flow cytometry.
Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.
Benschop
R J
RJ
University Hospital, Department of Immunology, Utrecht, The Netherlands.
de Smet
M B
MB
Bloem
A C
AC
Ballieux
R E
RE
D015703:Antigens, CD / Q000494:pharmacology; D018821:CD18 Antigens; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000494:pharmacology; D002860:Chromium Radioisotopes; D004727:Endothelium / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1; D007963:Leukocytes, Mononuclear / Q000502:physiology*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000494:pharmacology; D008214:Lymphocytes / Q000502:physiology; D009000:Monocytes / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015703:Antigens, CD; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D002860:Chromium Radioisotopes; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1; D013755:Tetradecanoylphorbol Acetate