Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes

• Published in: Journal of immunological methods 2000-05, Vol.239 (1), p.109-119
• Main Authors: Abbitt, Katherine B, Rainger, G.Ed, Nash, Gerard B
• Format: Article
• Language: English
• Subjects: Acridine Orange - pharmacology; Adhesion; Adult; Biological and medical sciences; Blood Platelets - physiology; CD18 Antigens - metabolism; Cell Adhesion - drug effects; Cell Movement - drug effects; Cell Movement - physiology; Cells, Cultured; Diverse techniques; Endothelium, Vascular - cytology; Fluoresceins - pharmacology; fluorescent dyes; Fluorescent Dyes - pharmacology; Fundamental and applied biological sciences. Psychology; Humans; integrins; Leukocytes, Mononuclear - drug effects; Leukocytes, Mononuclear - physiology; Molecular and cellular biology; mononuclear cells; neutrophils; Neutrophils - drug effects; Neutrophils - physiology; P-Selectin - metabolism; rheology; Rhodamines - pharmacology; selectins; Tumor Necrosis Factor-alpha - metabolism; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(00)00189-7

Fluorescent dyes assist visualisation of leukocytes for intravital studies of adhesion or for in vitro studies utilising whole blood. We have used in vitro flow-based assays to investigate the effects of three fluorescent dyes (acridine orange, AO, 5–100 μg/ml; calcein-AM, C-AM, 5–20 μg/ml; rhodamine 6G, R6G, 10–100 μg/ml) on adhesion and migration of isolated neutrophils and mononuclear cells. AO had little effect on the number or velocity of neutrophils rolling on P-selectin presented by a surface coated with platelets. However, AO did cause a dose- and time-dependent conversion of rolling to immobilisation. Pretreatment of neutrophils with an antibody against CD18 prevented this conversion to stationary adhesion, indicating that β 2 integrins were activated by AO. C-AM had little effect on neutrophil behaviour, but tended to cause some immobilisation at the highest concentration. R6G did not affect the number of neutrophils that bound to the platelet monolayer or the percentage rolling, but the rolling velocity of the neutrophils was increased in a dose-dependent manner. None of the dyes impaired the ability of neutrophils to respond to formyl peptide by converting from rolling to stationary adhesion. Neither C-AM nor R-6G reduced the number of flowing neutrophils or mononuclear cells binding to endothelial cells stimulated with tumour necrosis factor. Interestingly, R-6G inhibited transendothelial migration of mononuclear cells but not neutrophils, while C-AM did not affect transmigration of either cell type. The dose-dependent effects of dyes should be taken into consideration when designing any experimental protocol. AO does not appear to be a suitable dye for adhesion studies. R6G and C-AM can be used at ∼10 μg/ml (a concentration at which cells can be clearly visualised) although R-6G specifically inhibits the migratory response of mononuclear cells.