Flow cytometry analysis emerges as an alternative methodology to microscopy for determination of the Leishmania-infection rates of macrophages. Various flow cytometric approaches have been established for the quantification of Leishmania parasites within host cells, labelled either directly fluorescent dyes or indirectly with fluorescently conjugated antibodies. Although these techniques allow accurate quantification of infection, they fail at detection of non-infected macrophages specifically. This study introduces a new flow cytometric approach for the determination of infection rates of macrophages infected by Leishmania infantum parasites. Prior to infection, J774A.1 macrophages and L. infantum promastigotes were stained separately with PKH26 and PKH67 dyes, respectively. Dual staining enabled detection of each cell type, where non-infected macrophages were also recorded for the quantification. Dual-PKH staining achieved high success in selective staining of promastigotes (99.71%) and macrophages (99.57%). The percentages of parasite-infected macrophages were determined for initial 1:2.5 and 1:10 infection ratios as 15.68 and 61.70%, respectively; indicating significant increase in infection rate parallel to the initial treatment ratio. These results demonstrated that the introduced dual-fluorescence flow cytometric approach can be successfully used as an accurate and rapid quantification method for L. infantum-infected macrophages and strengthens the hypothesis that flow cytometric approaches could replace conventional microscopic methodologies.
Keywords: Detection of Leishmania infection; Leishmania infantum; PKH dyes; flow cytometry; fluorescent assay; macrophages.