title: Flow cytometric assessment of Leishmania spp metacyclic differentiation: validation by morphological features and specific markers.
abstract: Characterization of infective metacyclic promastigotes of Leishmania spp can be an essential step in several experimental protocols. Metacyclic forms of all Leishmania species display a typical morphology with short, narrow cell body, and an elongated flagellum. This feature suggests that metacyclics can be distinguished from procyclic forms by non-fluorimetric flow cytometric parameters thus enabling the follow-up of their appearance and acquisition of specific properties, during metacyclogenesis in in vitro cultures. Here we describe the flow cytometric parameters of stage-specific promastigotes of Leishmania major, Leishmania donovani, Leishmania amazonensis, and Leishmania braziliensis. Our findings were validated by optical microscopy morphology and specific procyclic labeling with FITC-peanut agglutinin. Furthermore, we show that parasite's distribution in the plot during differentiation in culture is not species specific and that the parasites displaying low forward-angle light scatter (FSC(low)) are three times more infective than the FSC(high) ones. The method here described can be applied to the identification of metacyclics of different Leishmania spp within the whole stationary population.
authors.affiliation: Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Elvira M
authors.initials: EM
authors.lastname: Saraiva
authors.forename: Lucia H
authors.initials: LH
authors.lastname: Pinto-da-Silva
authors.forename: João Luiz M
authors.initials: JL
authors.lastname: Wanderley
authors.forename: Adriana C
authors.initials: AC
authors.lastname: Bonomo
authors.forename: Marcello A
authors.initials: MA
authors.lastname: Barcinski
authors.forename: Maria Elisabete C
authors.initials: ME
authors.lastname: Moreira
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D007891:Leishmania / Q000254:growth & development* / Q000302:isolation & purification / Q000648:ultrastructure; D007892:Leishmania braziliensis / Q000254:growth & development / Q000302:isolation & purification / Q000648:ultrastructure; D007893:Leishmania donovani / Q000254:growth & development / Q000302:isolation & purification / Q000648:ultrastructure; D018320:Leishmania major / Q000254:growth & development / Q000302:isolation & purification / Q000648:ultrastructure; D007894:Leishmania mexicana / Q000254:growth & development / Q000302:isolation & purification / Q000648:ultrastructure; D008018:Life Cycle Stages; D017737:Macrophages, Peritoneal / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results; D013045:Species Specificity