A simple quantitative fluorimetric assay of in vitro phagocytosis in human neutrophils.
In general the in vitro assays of phagocytosis rely on microscope counting or radioisotopic detection of ingested particles or microbiological counting of non-ingested bacteria. A very simple, rapid, highly quantitative method using fluorescein-labelled bacteria was described by Vray et al. (Scand. J. Immunol. (1980) 11, 147) for non-human phagocytes. We report here a modification of this method to increase its sensitivity, to make it more suitable for pharmacological studies. We also provide detailed experimental parameters for its use with human phagocytes. A suspension of fluorescein-labelled bacteria is incubated with human phagocytes; after incubation at 37 degrees C, the reaction is terminated with ice-cold Tyrode buffer solution, and the non-ingested bacteria are removed by lysis with lysozyme and the resultant cell suspension treated with the detergent TX-100. The fluorescence of the suspension is then measured. The modified method is sufficiently sensitive to permit the detection of bi-directional effects on phagocytosis of a known modulator of human phagocyte function.
D001310:Auranofin / Q000494:pharmacology; D001770:Blood Bactericidal Activity; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D007700:Kinetics; D007958:Leukocyte Count; D008837:Micrococcus / Q000502:physiology; D009504:Neutrophils / Q000187:drug effects / Q000382:microbiology* / Q000502:physiology; D017830:Octoxynol; D010587:Phagocytosis / Q000187:drug effects; D011092:Polyethylene Glycols / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D005452:Fluoresceins; D013861:Thiocyanates; D001310:Auranofin; D011092:Polyethylene Glycols; D017830:Octoxynol; D016650:Fluorescein-5-isothiocyanate