Cell biotinylation provides a sensitive and effective detection technique for cellular adhesion assays: comparison with existing methods.
A simple, sensitive, colorimetric labelling method was devised to quantify cell adhesion, based on labelling the cell plasma membrane with biotin. This method was applied in adhesion assays, which involved the adherence of biotin-labelled, PMA-stimulated, U937 cells. These cells resemble monocytes, and were bound onto fibronectin-coated wells and to an ECV304 cell monolayer. The adherent U937 cells were detected by the addition of a peroxidase-conjugated anti-biotin antibody and a soluble colorimetric substrate. This assay is convenient, fast and sensitive, and able to detect 320-1000 U937 cells under the conditions described. This study has used titration assays to compare the biotinylation method with the existing cell quantification approaches of 51Cr radiolabelling and antibody dependent ELISA. Chromium labelling was the most sensitive technique, but we found the biotinylation method to be more convenient than radioactive labelling and more sensitive than conventional ELISA. Biotinylated cells were also used very effectively in a Stamper-Woodruff adhesion assay with U937 cells binding to histological sections of atherosclerotic plaques. The selective detection of the bound cells permitted automated quantitation by image analysis. Whole cell biotinylation may have wider applications in biological research.
Mendis
D
D
Centre for Cardiovascular Biology and Medicine, New Hunt's House, King's College London, Guy's Hospital Campus, SE1 1UL, London, UK.
Ginon
I
I
Louis
H
H
McGregor
J L
JL
Poston
R N
RN
D000911:Antibodies, Monoclonal / Q000276:immunology; D019921:Biotinylation / Q000379:methods*; D002340:Carotid Artery Diseases / Q000473:pathology*; D002448:Cell Adhesion; D002460:Cell Line; D002860:Chromium Radioisotopes; D003124:Colorimetry / Q000379:methods*; D004727:Endothelium / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000276:immunology*; D009000:Monocytes / Q000276:immunology; D012680:Sensitivity and Specificity; D020298:U937 Cells
D000911:Antibodies, Monoclonal; D002860:Chromium Radioisotopes; D005353:Fibronectins