Measurement of phagocytosis using fluorescent latex beads

Fluorescent monodisperse latex beads and a computer-centered spectrofluorimeter were used to devise a sensitive new assay for phagocytosis. LM fibroblasts, a transformed cell line with a high endocytic rate, were exposed to fluoresbrite beads and the following parameters were investigated: incubation time, incubation temperature and bead/cell ratio. The bead uptake was linear for 60 min over a wide range of bead/cell ratios up to 130 beads/cell. Phagocytosis was inhibited at 4 degrees C, by incubation in the presence of colchicine, and by glucose deprivation. Scanning and transmission electron microscopy were used to confirm that at 37 degrees C both bead adsorption and internalization occurred while at 4 degrees C only bead adsorption but not endocytosis occurred. Large bead sizes (0.86 and 1.72 micrometer diameter) were most useful due to higher fluorescence and higher signal to noise ratios than smaller beads (0.25 and 0.57 micrometer diameter). Beads (0.86 micrometer diameter) were taken up at a rate of 4.4 beads/cell/h at 37 degrees C when a bead/cell ratio of 70 was used. The uptake was zero when assayed at zero time. These criteria establish that fluoresbrite beads provide a useful new fluorimetric assay for phagocytosis.