Abstract
Background:
Macrophage phagocytosis is a fundamental component of innate immunity in animals and plays a critical role in host defense against bacterial infection, inflammation, and tissue homeostasis. In vitro macrophage assays are widely used in veterinary immunology research. The biochemical and biophysical conditions used in the culturing of cells is known to influence macrophage phagocytosis. However, the effect of the surface coating substrate of culture dishes on phagocytic activity in vitro remains unclear.

Aim:
This study aimed to evaluate how commonly used glass surface coatings affect macrophage phagocytic activity and to identify an optimal in vitro culture condition suitable for veterinary immunology research.

Methods:
Non-coated (NC), poly-L-lysine-coated (PL), and collagen-coated (CC) glass-bottom dishes were compared as culture surface coating substrates for RAW264.7 macrophages. Lipopolysaccharide-stimulated RAW264.7 cells were treated with fluorescent latex beads and phagocytic activity was subsequently quantified by assessing the proportion of bead-positive cells, intracellular bead load, and composite phagocytic index after 3 h and 24 h.

Results:
All surface coating substrates supported similar adhesion and comparable phagocytic performance at 3 h. However, at 24 h the cells cultured on NC dishes showed a significantly higher proportion of phagocytic activity, greater bead internalization per cell, and the highest phagocytic index by comparison to those cultured on PL or CC dishes.

Conclusion:
These findings demonstrate that the culture surface coating significantly influences macrophage phagocytic activity during prolonged incubation. NC glass provides the most appropriate and least confounding substrate for in vitro macrophage phagocytosis assays and should be preferentially considered in veterinary immunology.

Key words: Macrophage; Phagocytosis; RAW264.7; Surface coating substrate; Veterinary immunology.