Effects of cell culture density on phagocytosis parameters in IC-21 macrophages

Cell culture density is shown to alter the parameters characterizing phagocytic activity of cells in vitro. Phagocytosis index (PI, mean number of beads per cell in the bead-containing population) and phagocytosis percent (PP, percentage of bead-containing cells in cell population under study) for IC-21 macrophages incubated in the presence of non-opsonized 2-μm fluorescent latex beads were determined using fluorescent microscopy and ImageJ software specially adapted for the purpose. Under control conditions (DMEM without serum), increase in cell culture density was accompanied with a decrease of both parameters of the phagocytic activity. At a mean density of 4 cells/10⁵ μm² (9 cells per a viewfield) PI was 7.1 ± 0.2 beads/cell and at 20 cells/10⁵ μm² (40 cells per a viewfield) PI dropped to 4.6 ± 0.1 beads/cell. PP was less sensitive, varied in the range of 95–100% but also decreased as the cell density grew. At any density, PI was 1.5–2 times higher than the expected value (number of beads per μm² × cell contour area); apparently this divergence can be accounted for by cell locomotion and capture of a larger number of beads than could drop onto a motionless cell with a constant contour area. Increase in cell density was also accompanied by a decrease of the cell contour area (S _c), which amounted to 750 ± 16 μm² at a density of 4 cells/10⁵ μm² and 346 ± 4 μm² at a density of 20 cells/10⁵ μm². As the bead concentration was the same in all experiments, density-dependent decrease in PI and PP may be related with the observed decrease in cell contour area. Yet, the bead number per cell area unit (PI/S _c) was bigger at higher density and PI/S _c was higher in cells with smaller S _c. Thus, individual (specific) activity of the cells did not lessen with an increase of the cell culture density in the range studied (4–20 cells/10⁵ μm²). Reduction of the cell contour area may reflect alteration in cell adhesion to the substrate as well as competitive relations between adhesion and phagocytic processes. The data obtained imply that cell culture density has to be controlled as a factor notably altering the phagocytic activity parameters. The effects of serum, methyl-β-cyclodextrin, and carbenoxolon reported earlier [Golovkina et al. 2009. Biol membrany. 26 (5), 379–386] are re-evaluated and confirmed here.