Accurate quantitation of Leishmania infection in cultured cells by flow cytometry

Background: Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of th eir Life cycle. Quantitation of experimental mammalian cell infections is u sually performed by time-consuming microscopic examination. In this report a flow cytometry (RCM)-based assay suitable for studying in vitro infection s by L. amazonensis is presented. Methods: Intense fluorescence staining of the amastigote forms with a stage - and species-specific monoclonal antibody was obtained after permeabilizat ion of both the host-cell cytoplasmic membrane and the parasitophorous vacu ole membrane by saponin treatment. Results: Upon flow cytometry (FCM) analysis, parasitized cells separated sh arply from the auto-fluorescence of the mammalian host cells, giving the as say a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contra st and UV-light microscopy, while no para-sites were observed in more than 35% of the cells in the population with background fluorescence. Comparison s of the FCM results with those from microscope counting and analysis of va rious dilutions of parasitized cells con firmed the reliability of the meth od. Conclusions: The FCM assay provided rapid quantitation of Leishmania infect ion either in mouse macrophages, the natural host cell in murine leishmania sis, or in Chinese hamster orary (CHO) cells, a non-macrophage cell line pr oposed as an in vitro model for studying host-parasite interactions. The pr otocol described here should be adaptable to studies involving other parasi tes residing in nucleated cells. (C) 2000 Wiley-Liss, Inc.