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

ABSTRACT: 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 ?g/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.

SUBMITTER: Yektaeian N

PROVIDER: S-EPMC6928280 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Lipophilic tracer Dil and fluorescence labeling of acridine orange used for <i>Leishmania major</i> tracing in the fibroblast cells.

Yektaeian Narjes N Mehrabani Davood D Sepaskhah Mozhdeh M Zare Shahrokh S Jamhiri Iman I Hatam Gholamreza G

Heliyon 20191218 12

<h4>Background</h4>This study aims to evaluate the use of fluorescent dye Dil and super vital dye acridine orange (AO) <i>in vitro</i> tracking of labeled <i>L. major</i> in the fibroblast cells.<h4>Methods</h4>Dil crystal and AO were used to stain <i>L. major</i> in a co-culture of the fibroblasts with the parasite. AO staining solution was added to 1 × 10<sup>6</sup> parasites. After 10 min, the stained parasites were centrifuged and washed seven times with phosphate buffered saline (PBS). The ...[more]

PMID: 31890980

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Project description:BackgroundViral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK.MethodsUsing our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gKwt) or the mutant gK (gKmut, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gKmut pseudotyped and gKwt coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties.ResultsH129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gKmut pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gKwt, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer's disease and control mouse brains.ConclusionsgKmut pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.

Dataset Information

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

Publications

Lipophilic tracer Dil and fluorescence labeling of acridine orange used for <i>Leishmania major</i> tracing in the fibroblast cells.

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