NBT-coated yeast cell-phagocytosis test, bactericidal activity, and chemotaxis assay. (A) NBT-coated yeast cells were added to a neutrophil suspension and incubated at 37°C. After 1 hour, the cells were stained with 1% safranin-O, and observed using a microscope. Mature neutrophils () could be easily distinguished from contaminating macrophages (white arrow; only the nucleus is observed in the figure) by the unique shape of their nuclei. Yeast cells were red-brown in color before being ingested (white arrowhead); the color began to change to purple or black beginning at the periphery of the yeast cell, and eventually became completely black () because the NBT was reduced after ingestion. Yeast cells that changed color in the cells were counted as NBT-reduction positive. Original magnification, ×400. (B) The phagocytosis rate was calculated as a percentage of the neutrophils that contained one or more yeast cells. hESC-Neu's had a slightly lower phagocytosis rate than that of PB-Neu(G−)'s and PB-Neu(G+)'s. (C) The phagocytosis score was calculated as the total number of positive yeast cells in 100 neutrophils. There were no significant differences in the phagocytosis score between hESC-Neu's and PB-Neu(G−)'s or PB-Neu(G+)'s. The cells on day 8 of the culture (day-8 cells) were rarely observed to phagocytose the yeast cells or reduce NBT. (In B-C, n = 3; bars indicate SDs; *P < .05 compared with PB-Neu(G−)'s and PB-Neu(G+)'s; **P < .05 compared with hESC-Neu's, PB-Neu(G−)'s, and PB-Neu(G+)'s.) (D) Bactericidal assay. E coli was opsonized with human AB serum, and incubated with hESC-Neu's, PB-Neu(G−)'s, PB-Neu(G+)'s, or control medium. After 1-hour incubation with hESC-Neu's, PB-Neu(G−)'s, and PB-Neu(G+)'s, the colony-forming units (CFUs) were significantly reduced to approximately 40% of the control. There were no significant differences in bactericidal activity between hESC-Neu's, PB-Neu(G−)'s, and PB-Neu(G+)'s. The CFUs of controls are presented as 100% (n = 3; bars indicate SDs; *P < .05 compared with control). (E) Chemotaxis assay by a modified Boyden chamber method. The number of neutrophils that migrated randomly (fMLP(−)) was not significantly different between hESC-Neu's and PB-Neu(G−)'s, but PB-Neu(G+)'s showed significantly greater random migration than hESC-Neu's and PB-Neu(G−)'s. The number of migrating cells increased in all hESC-Neu's, PB-Neu(G−)'s, and PB-Neu(G+)'s when fMLP was added to the lower well (fMLP(+)). The increase in the number of migrating cells induced by chemotaxis to fMLP (fMLP(+)-fMLP(−)) was not significantly different between hESC-Neu's and PB-Neu(G−)'s or PB-Neu(G+)'s (n = 3; bars indicate SDs; *P < .05).