A rapid and simple microfluorometric phagocytosis assay.
A microfluorometric method for phagocytosis study has been developed using fluorescein conjugated Escherichia coli K-12 particles. This technique is based on the uptake of fluorescent particles and quenching of extracellular fluorescence at the end of the assay. A murine macrophage cell line, J774, was used as a phagocyte model. The cells were harvested from tissue culture flasks and adjusted to 1 x 10(6) cells/ml. They were then dispensed into a 96-well tissue culture plate, 100 microliters/well, 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 and 100 microliters of fluorescent E. coli particles suspended in Hanks' buffer were added. The plates were further incubated for various time periods. Buffer solution in the wells was removed by aspiration. Extracellular fluorescence was then quenched by adding 100 microliters of trypan blue (250 micrograms/ml, pH 4.4). The dye was removed after 1 min. The intensity of fluorescence associated with intracellular fluorescent particles was measured directly in the wells using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. This assay provided a rapid and objective measurement of phagocytosis activity. Using a cultured cell line and a 96-well microtiter plate format, this assay can facilitate the screening of a large number of various biological and pharmacological substances for their modulating effects on phagocytosis.
Wan
C P
CP
Department of Microbiology, School of Medicine, Loma Linda University, CA 92350.
Park
C S
CS
Lau
B H
BH
D000818:Animals; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D003592:Cytophotometry / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D019793:Fluorescein; D005452:Fluoresceins; D008264:Macrophages; D051379:Mice; D010587:Phagocytosis / Q000187:drug effects; D015203:Reproducibility of Results; D014343:Trypan Blue
D005452:Fluoresceins; D015638:Cytochalasin D; D014343:Trypan Blue; D019793:Fluorescein