Phagocytosis: quantification of rates and intercellular heterogeneity

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Abstract
We present a method which permits analysis of phagocytic behavior in small samples of macrophages. Both overall phagocytic kinetics and intercellular variability in particle uptake were measured. Macrophages lavaged from Syrian golden hamster lungs were incubated (5 min at 37 degrees C) with 2- to 4-mum plastic particles at 4.6, 6.9, 9.1, and 16.0 X 10(6) part./ml. Harvested macrophages ranged from 0.6 to 4.0 X 10(6) cells per animal. To concentrate the cells and separate them from cell-free substrate after termination of phagocytosis, 30–60% of each flask's contents were centrifuged (400 X g, 20 min) atop a Metrizamide subphase (34% wt/vol, rho = 1.18). Cells were collected from the interface, fixed, and visually scored for number of particles phagocytized. Phagocytic rates followed Michaelis-Menten kinetics with Vmax = 0.63 +/- 0.18 (SEM) part./viable cell per min and Km = 8.5 +/- 2.7 (SEM) X 10(6) part./ml. In every case (23/23 flasks from 6 animals) particle uptake exhibited greater (P less than 0.01) cell-to-cell variability in avidity for substrate than the Poisson distribution would predict from mean number of particles phagocytized per viable cell.

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