Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes.
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 microg/ml; calcein-AM, C-AM, 5-20 microg/ml; rhodamine 6G, R6G, 10-100 microg/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 beta(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 approximately 10 microg/ml (a concentration at which cells can be clearly visualised) although R-6G specifically inhibits the migratory response of mononuclear cells.
Abbitt
K B
KB
Department of Physiology, Division of Medical Science, The Medical School, The University of Birmingham, B15 2TT, Birmingham, UK.
Rainger
G E
GE
Nash
G B
GB
D000165:Acridine Orange / Q000494:pharmacology*; D000328:Adult; D001792:Blood Platelets / Q000502:physiology; D018821:CD18 Antigens / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000502:physiology; D009504:Neutrophils / Q000187:drug effects* / Q000502:physiology; D019007:P-Selectin / Q000378:metabolism*; D012235:Rhodamines / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology
D018821:CD18 Antigens; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019007:P-Selectin; D012235:Rhodamines; D014409:Tumor Necrosis Factor-alpha; C026188:rhodamine 6G; C085925:calcein AM; D000165:Acridine Orange