Flow cytometry technique for analysingLeishmaniapromastigote phagocytosis by human polymorphonuclear leucocytes and monocytes

Abstract

In this study, we developed a flow cytometry technique for studying Leishmania (L.) mexicana phagocytosis by human polymorphonuclear leucocytes (PMNs) and monocytes. Leishmania promastigotes are elongated in shape and flagellated. This influences the light scatter when phagocytosis is measured by flow cytometry. Accordingly, we developed an oxidative burst method for measuring the phagocytic process. As this is an indirect marker of phagocytosis, we used confocal, light and electron microscopy to verify that promastigotes were, indeed, internalized by the phagocytes. For both PMNs and monocytes, the optimal conditions for achieving high sensitivity in flow cytometry detection were 5% pooled human serum and 15 min. incubation time. Incubations at 35, 37 and 39°C were also equally efficient for both PMNs and monocytes. Optimal parasite ratios were 10 parasites per PMN and 20 parasites per monocyte. Under these conditions, Leishmania were readily phagocytosed by human PMNs and monocytes and the effects of other influences, such as treatment, would be readily detectable. This indicated that these cells may play a role in the immune response against Leishmania.