FIG. 7.

Staining of viable intracellular L. major in PMN with ethidium bromide and acridine orange. PMN were coincubated with L. major promastigotes at a PMN-to-parasite ratio of 1:5 for 3 h at 37°C in RPMI 1640 medium supplemented with 20% heat-inactivated FCS, separated from nonphagocytosed L. major promastigotes, and subsequently incubated for 24 h at 37°C. The viability of L. major in PMN was then assessed by staining with acridine orange to visualize viable cells or parasites (green staining) and ethidium bromide, which stains dead cells. Viable L. major parasites (arrows) are seen in nonapoptotic viable PMN. The morphological appearance of the nuclei and kinetoplasts is typical for intact parasites.