Back to the Basics: Two Approaches for the Identification and Extraction of Lipid Droplets from Malassezia pachydermatis CBS1879 and Malassezia globosa CBS7966
Mantilla, Maria Juliana; Cabrera Díaz, Catherine Eliana; Ariza-Aranguren, Gabriela; de Cock, Hans; Helms, J. Bernd; Restrepo, Silvia; Jiménez, Elizabeth; Celis Ramírez, Adriana Marcela
(2021) Current Protocols, volume 1, issue 5, pp. 1 - 16
(Article)
Abstract
Malassezia spp. are lipid-dependent yeasts that have been related to skin mycobiota and dermatological and systemic diseases. Study of lipid droplets (LDs) is relevant to elucidate the unknown role of these organelles in Malassezia and to gain a broader overview of lipid metabolism in Malassezia. Here, we standardized two protocols
... read more
for the analysis of LDs in M. pachydermatis and M. globosa. The first describes co-staining for confocal laser-scanning fluorescence microscopy, and the second details extraction and purification of LDs. The double stain is achieved with three different neutral lipid fluorophores, namely Nile Red, BODIPY™ 493/503, and HCS LipidTOX™ Deep Red Neutral, in combination with Calcofluor White. For LD extraction, cell wall rupture is conducted using Trichoderma harzianum enzymes and cycles of vortexing with zirconium beads. LD purification is performed in a three-step ultracentrifugation process. These standardizations will contribute to the study of the dynamics, morphology, and composition of LDs in Malassezia.
show less
Download/Full Text
Keywords: cell wall rupture, fluorescence microscopy, lipid droplets, Malassezia globosa, Malassezia pachydermatis, Taverne, Biochemistry, Genetics and Molecular Biology(all), Pharmacology, Toxicology and Pharmaceutics(all), Neuroscience(all), Immunology and Microbiology(all), Health Informatics, Medical Laboratory Technology, Medicine(all)
ISSN: 2691-1299
Publisher: Wiley
Note: Funding Information: This work was supported by the Faculty of Sciences, Universidad de Los Andes, grant no. INV‐2018‐31‐1252, Project Semilla INV‐2018‐49‐1371, Project FAPA‐P17160322012, and the Vice‐Presidency of Research and Creation. We thank μ‐core at Universidad de Los Andes ( https://microcore.uniandes.edu.co/es/ ). Publisher Copyright: © 2021 Wiley Periodicals LLC
(Peer reviewed)
See more statistics about this item