Superoxide production of hESC-Neu's assessed by dihydrorhodamine123 oxidation. (A) Dihydrorhodamine123 (DHR) was reacted to neutrophils with or without phorbol myristate acetate (PMA), and the resultant rhodamine fluorescence was detected by flow cytometry. When DHR was added to the reaction medium without PMA (line), the fluorescence levels were slightly elevated in hESC-Neu's (i), PB-Neu(G−)'s (ii), and PB-Neu(G+)'s (iii). The addition of PMA dramatically increased the levels of fluorescence in all 3 neutrophil preparations (bold line). The figures are representative of 3 independent experiments. (B) Comparison of superoxide production between hESC-Neu's and PB-Neu's using mean fluorescence intensity (MFI) of rhodamine. When DHR was added without PMA stimulation, rhodamine-specific fluorescence was detected in hESC-Neu's, PB-Neu(G−)'s, and PB-Neu(G+)'s. PMA stimulation increased rhodamine MFI in hESC-Neu's though to a lesser extent than in PB-Neu(G−)'s and PB-Neu(G+)'s. Consequently, rhodamine MFI after PMA stimulation was similar in hESC-Neu's, PB-Neu(G−)'s, and PB-Neu(G+)'s, suggesting that the maximum superoxide production was comparable between hESC-Neu's and PB-Neu's (n = 3; bars represent SDs; *P < .05 compared with hESC-Neu's).