5] Oil-droplet method for measuring phagocytosis

Abstract

Publisher Summary The basis of the oil-droplet method to assay phagocytosis is to stabilize particles containing a marker dye or radioactive compound, feed them to phagocytes, separate uningested particles from the cells by differential centrifugation, extract the marker from the cells, and measure it. The rate of association of the marker with the centrifuged cell pellet as a function of time reflects the rate of phagocytosis. Since its initial description, the oil-droplet method has been modified in many ways. Originally, the dye oil red O was incorporated as a marker into heavy paraffin oil. Subsequently, di-isodecyl phthalate was found to have a more suitable density than paraffin oil. Other modifications have included the use of radioactive lipids as markers and of spin-labeled cholestanone, which can be quenched by ascorbic acid, permitting assessment of the extent to which particles are either adherent to the cells or are incorporated within unsealed vacuoles. Double-label experiments have been performed by feeding cells particles labeled with either oil red O or perylene, measuring the former spectrophotometrically and the latter fluorimetrically. This method has several advantages, the most important of which is the fact that the density of suitable oil droplets is such that the efficiency of separation of uningested particles from phagocytes is very high. The ability to remove adherent particles markedly facilitates the precise quantitation of phagocytic rates. One disadvantage of the assay is that the particles, unlike erythrocytes, zymosan, or latex, cannot be simply purchased as such but must be prepared by the investigator.