A Fluorescence-Based Method To Visualize Lipid Droplet And Organelle Dynamics During Mitosis in Hepatoma Cells

Intracellular lipid droplets (LDs) are ubiquitous in several cell types. LDs store neutral lipids such as triacylglycerol and cholesteryl ester and play important roles in energy metabolism, signaling, and stress responses. Although LDs are recognized as intracellular organelles, their behavior during cell cycle progression remains unclear. During mitosis, several organelles, including mitochondria and the Golgi apparatus, undergo extensive remodeling and subsequent reassembly, raising the question whether LDs also undergo cell cycle-dependent changes. Since LDs can serve as energy sources and signaling hubs, clarifying their dynamics during mitosis is of particular importance. To address this, we present a protocol that combines cell cycle synchronization with fluorescent LD staining in hepatoma cells. Cells are synchronized at the G2/M boundary using a cyclin-dependent kinase 1 inhibitor-based approach, released into the cell cycle, and collected at specific time points. The LDs are then labeled with a neutral lipid dye and visualized by fluorescence microscopy. This method provides a reproducible approach for examining LD redistribution during mitosis and can be applied broadly to studies of organelle biology, metabolic regulation, and cancer cell physiology.