A RAPID AND SIMPLE MICROFLUOROMETRIC PHAGOCYTOSIS ASSAY

A microfluorometric method for phagocytosis study has been developed u sing fluorescein conjugated Escherichia coli K-12 particles. This tech nique is based on the uptake of fluorescent particles and quenching of extracellular fluorescence at the end of the assay. A murine macropha ge cell line, J774, was used as a phagocyte model. The cells were harv ested from tissue culture flasks and adjusted to 1 x 10(6) cells/ml. T hey were then dispensed into a 96-well tissue culture plate, 100 mul/w ell, and incubated at 37-degrees-C in 5% CO2 for 1 h to allow cells to adhere to the bottom of the wells. The culture medium was aspirated a nd 100 mul of fluorescent E. coli particles suspended in Hanks' buffer were added. The plates were further incubated for various time period s. Buffer solution in the wells was removed by aspiration. Extracellul ar fluorescence was then quenched by adding 100 mul of trypan blue (25 0 mug/ml, pH 4.4). The dye was removed after 1 min. The intensity of f luorescence associated with intracellular fluorescent particles was me asured directly in the wells using a computerized microplate fluoromet er at 485 nm excitation and 530 nm emission. This assay provided a rap id and objective measurement of phagocytosis activity. Using a culture d cell line and a 96-well microtiter plate format, this assay can faci litate the screening of a large number of various biological and pharm acological substances for their modulating effects on phagocytosis.