A Protocol for Size-Based Enrichment of Lipid Droplets from Bovine Mammary Epithelial Cells and Mammary Tissues

This protocol describes a sequential differential centrifugation approach to enrich lipid droplets (LDs) of different sizes from bovine mammary epithelial cells (BMECs) and mammary tissues, enabling size-resolved analysis beyond conventional bulk LD isolation methods. As dynamic regulatory hubs of intracellular lipid metabolism, LDs serve critical functions by storing neutral lipids (such as triglycerides and cholesteryl esters) and coordinating their synthesis, hydrolysis, and transport. They play a central role in maintaining energy homeostasis, supporting membrane biogenesis, and facilitating cellular signal transduction. LD extraction is essential for investigating the regulatory mechanisms of lipid metabolism. Studying mammary gland lipid metabolism has significant implications for infant development, human health, agricultural economics, and fundamental cell biology. Therefore, we describe a differential centrifugation method for extracting LDs from BMECs and mammary gland tissue. In contrast to conventional bulk LD isolation approaches, this protocol enables size-based enrichment of LD subpopulations by sequentially adjusting centrifugal forces. The integrity and relative enrichment of LD fractions are preliminarily evaluated using BODIPY493/503 staining, providing a practical and reproducible approach for downstream analyses of lipid metabolism and LD-associated processes.