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Protocol for observing lipid droplet dynamics in chicken cone cells.

ABSTRACT: Chicken cone cells are an excellent model for studying the regulation of lipid droplet dynamics. Here, we present a protocol for studying cone cell lipid droplets from in vivo and ex vitro cultured retinas of chicken embryos. We describe steps for dissecting chicken retinas, electroporating retinas, culturing retinas ex vivo and in vitro, and staining lipid droplets with neutral lipid dye. This protocol is also applicable to investigating other organelles in retinas. For complete details on the use and execution of this protocol, please refer to Pan et al.¹.

SUBMITTER: Pan H

PROVIDER: S-EPMC11216057 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature

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Protocol for observing lipid droplet dynamics in chicken cone cells.

Pan Huimin H Zhu Jinglin J Huang Xun X

STAR protocols 20240605 2

Chicken cone cells are an excellent model for studying the regulation of lipid droplet dynamics. Here, we present a protocol for studying cone cell lipid droplets from in vivo and ex vitro cultured retinas of chicken embryos. We describe steps for dissecting chicken retinas, electroporating retinas, culturing retinas ex vivo and in vitro, and staining lipid droplets with neutral lipid dye. This protocol is also applicable to investigating other organelles in retinas. For complete details on the ...[more]

PMID: 38843400

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Project description:BackgroundAccumulation of lipid droplets (LDs) was recently observed in pyometra-affected uteri. As data about their nature and function are missing we intended to compare the localization, quality and quantity of LDs in canine healthy and pyometra-affected tissues and in an in vitro model.Methods and resultsWe characterized LDs in healthy and pyometra uterine tissue samples as well as in canine endometrial epithelial cells (CEECs) in vitro by means of histochemistry, immunohistochemistry, transmission electron microscopy, western blot, and RT-qPCR. Oil Red O (ORO) staining and quantification as well as p-phenylenediamine staining showed a higher number of LDs in epithelial cells of pyometra samples. Immunohistochemistry revealed that the amount of LDs coated by perilipin2 (PLIN2) protein was also higher in pyometra samples. Transmission electron microscopy showed an increase of LD size in surface and glandular epithelial cells of pyometra samples. In cell culture experiments with CEECs, supplementation with oleic acid alone or in combination with cholesterol lead to an increased LD accumulation. The expression of PLIN2 at protein and mRNA level was also higher upon oleic acid supplementation. Most LDs were double positive for ORO and PLIN2. However, ORO positive LDs lacking PLIN2 coating or LDs positive for PLIN2 but containing a lipid class not detectable by ORO staining were identified.ConclusionsWe found differences in the healthy and pyometra-affected endometrium with respect to LDs size. Moreover, several kinds of LDs seem to be present in the canine endometrium. In vitro studies with CEECs could show their responsiveness to external lipids. Since epithelial cells reacted only to oleic acid stimulation, we assume that the cyclic lipid accumulation in the canine endometrium is based mainly on triglycerides and might serve as energy provision for the developing early embryo. Further studies are necessary to verify the complex role of lipids in the healthy and pyometra-affected canine endometrium.

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Protocol for observing lipid droplet dynamics in chicken cone cells.

Publications

Protocol for observing lipid droplet dynamics in chicken cone cells.

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