Lipophilic tracer Dil and fluorescence labeling of acridine orange used for Leishmania major tracing in the fibroblast cells

Background: This study aims to evaluate the use of Fluorescent Dye Dil and super vital dye acridine orange (AO) in vitro tracking of labeled L. major in the fibroblast cells.

Methods: Dil crystal and AO were used to stain L. major in a co-culture of the fibroblasts with the Parasite. AO staining solution was added to 1 × 10⁶ parasites. After 10 min, the stained parasites were centrifuged and washed seven times with phosphate buffered saline (PBS). The stained promastigote was incubated with fibroblasts for 6-8 h. The presence of stained parasites with AO in the fibroblast was assessed using a fluorescence microscope. 1 × 10⁶/mL promastigote of L. major was gently suspended and mixed by Dil staining solution with an ultimate concentration of 0.002 μg/mL and it was kept for 20 min at the room temperature. Subsequently, after washing it in PBS for seven times, it was centrifuged at 3000 rpm for 10 min. The supernatant was removed and the precipitate containing stained promastigote was suspended in fresh DMEM F12 with fibroblasts at 37 °C for 6 h. The presence of stained parasites with Dil in fibroblast was assessed using a fluorescence microscope. Fibroblast characterization was undertaken by a real-time polymerase chain reaction (PCR).

Results: Acridine orange staining assisted in detection of the live Parasite in the fibroblast cells. Free promastigote looked green before entering into the fibroblasts after 12 h culture. The Parasite entered the cytoplasm of fibroblasts at the beginning of the exposure and gradually entered the nucleus of the fibroblast. The fibroblast nucleus was entirely stained green by AO. The L. major appeared green under the fluorescent microscope. Dil staining revealed that the internalized parasites with red/orange color were localized within the cytoplasm after 6-hours and the nucleus of the fibroblasts after 72-hours following culture. Human fibroblasts were positive at the expression of CD10, CD26, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) and negative for CD106 and Integrin alpha 11.

Conclusion: The Fluorescent Dye Dil staining is a safe, easy to use, inexpensive and fast method for labeling of the Leishmania Parasite in the fibroblast cells. Acridine orange staining could be useful for tracing the parasites in the fibroblasts too. In this study, both Dil and AO were compared and considered as suitable vital dyes for identifying labeled Leishmania in the fibroblast in vitro, but Dil was superior to AO with its feature does not transfer from the labeled to unlabeled cells.