Flow cytometry in the study of the interaction between murine macrophages and the protozoan parasite Leishmania amazonensis.
A flow cytometry method was adapted to study interaction between murine macrophages and Leishmania amazonensis. Using this method it was possible to detect internalization of parasites through an increase in macrophage granularity (side scatter), with the latter indicating the presence of parasites inside parasitophorus vacuoles. A quenching technique was used to confirm the feasibility of the method and to distinguish between internalized and externally attached parasites. Experiments using fixed-labeled and killed-unlabeled parasites gave similar results, demonstrating that granularity was an adequate parameter in the study of parasite-macrophage interaction, when compared with labeling methods. Experiments that measured internalization using only the increase in macrophage granularity as an indicator showed that living L. amazonensis was internalized to a greater extent than killed-unlabeled parasites. This finding suggests that the parasite has an active role in the process of internalization. The 2 methods in combination, flow cytometry and labeling, can be used to study murine peritoneal cell-L. amazonensis interactions and to sort phagocytosing and nonphagocytosing subpopulations of macrophages for further studies.
Bertho
A L
AL
Department of Protozoology, Oswaldo Cruz Institute, Rio de Janeiro, Brazil.
Cysne
L
L
Coutinho
S G
SG
D000818:Animals; D002478:Cells, Cultured; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D007700:Kinetics; D007894:Leishmania mexicana / Q000502:physiology*; D008264:Macrophages / Q000469:parasitology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010529:Peritoneal Cavity / Q000166:cytology
D016650:Fluorescein-5-isothiocyanate