Flow Cytometry Optimization of Three Different BODIPY Molecules for Staining Lipid Droplets of Cryptococcus neoformans

Abstract

The pathogenic Cryptococcus neoformans causes life-threatening disease in immunocompromised patients. Although there are hypotheses about the role of lipid droplets in C.neoformans pathogenesis, there is still not extensively data determined on the subject yet. Lipid droplets are dynamic cytoplasmic energy storage bodies in yeasts. Diazo dyes, Nile red, LD 540 and borradiazaindasen (BODIPY) molecules are frequently used in the lipid droplets studies. BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyestuffs are among the brightest green light emitting fluorophores. These neutral molecules have high lipophilicity and can easily pass through the cell wall and membrane. In this study, for the future studies on lipid droplets of C.neoformans, we aimed to optimize three different BODIPY (BODIPY480/525, BODIPY480/530, BODIPY480/535) molecules for fluorescence microscopy and flow cytometry system. Ten molecularly confirmed environmental C.neoformans strains were grown on Sabouraud dextrose agar with and without oleic acid. BODIPY staining protocols at different concentrations were used for C.neoformans lipid droplets fixed with paraformaldehyde. The visualization (by fluorescence microscopy) and detection (by flow cytometer) of the lipid droplet structures of C.neoformans strains were evaluated. Forwardscatter and side-scatter analysis were performed to evaluate the number of lipid droplets determined in the cytoplasmic region quadrant in flow cytometry. The staining of the lipid droplets of C.neoformans of all three BODIPY molecules used in the study was observed by fluorescence microscope with creating distinct brightness and sharp contrast with the background. All BODIPY molecules could be examined by fluorescence microscopy without loss of brightness in more than one minute. The optimal dye concentration of BODIPY compounds were found as 2 mu M. Incubation at room temperature for five minutes was sufficient for fluorochrome staining. They were also shown to be able to stain lipid droplets in heatinactivated C.neoformans strains in all three compounds. The synthesized BODIPY480/525, BODIPY480/53 and BODIPY480/535 molecules were evaluated in accordance with the staining of lipid droplets, which were claimed to play a role in the pathogenesis of C.neoformans, and analysis by fluorescence microscopy and analysis with flow cytometer. BODIPY molecules may exhibit different properties in staining lipid droplets. These molecules should be tested for demonstrating the presence of lipid droplets in different yeast species and their suitability for pathogenesis studies.