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

Summary

This protocol enables reproducible visualization of lipid droplets and organelle dynamics during mitosis using synchronized hepatoma cells.

Abstract

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.

Introduction

The cell cycle is a highly orchestrated event that ensures the accurate duplication and segregation of chromosomal DNA^1,2, during which intracellular organelles are dynamically reorganized³. It consists of four major phases: G1, S, G2, and M^1,2. During mitosis, duplicated chromosomes align at the equatorial plane of the bipolar spindle and are subsequently transferred to two daughter cells⁴. Organelles such as mitochondria and the Golgi apparatus undergo extensive remodeling during mitosis

Protocol

1. Stock solutions required for this protocol

1. Prepare 24% (w/v) fatty acid–free bovine serum albumin (BSA) solution dissolved in 150 mM NaCl.
2. Prepare 10 mM RO-3306 (a selective cyclin-dependent kinase 1 (CDK1) inhibitor) stock solution dissolved in DMSO under sterile conditions.
   NOTE: Other commonly used solutions, such as NaOH, NaCl, and phosphate-buffered saline (PBS), were prepared according to standard laboratory practice. PBS was prepared by dissolving 2.6 g of NaH₂PO₄·2H₂O, 29 g of Na₂HPO₄·12H₂O, 80 g of NaCl, and 2.0 g of KCl in distilled water and adjusting the fi....

Results

Synchronization of hepatoma cells at the G2/M boundary using RO-3306

To investigate the dynamics of intracellular organelles during mitosis, RO-3306, a selective CDK1 inhibitor, was used to synchronize the cells at the G2/M boundary^20,21. When cells were treated with the CDK1 inhibitor for 20 h followed by an additional 1 h in a fresh medium, over 50% of the cells were synchronized at the G2/M boundary (

Discussion

The observed redistribution of LDs during mitosis suggests that their positioning is not random but is in coordination with cytoskeletal reorganization and cell cycle progression. In particular, the exclusion of LDs from the spindle region may reflect spatial constraints imposed by mitotic spindle assembly or active transport mechanisms that maintain organelle segregation during cell division. In this study, we present a simple and reproducible approach to prepare mitotic cells for immunofluorescence staining, while visu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin/EDTA	Wako	201-16945	Cell dissociation
10 cm dish	Corning	430167	Cell culture
35 mm tissue culture dish	IWAKI	3000-035	Cell culture
6 cm tissue culture dish	IWAKI	3010-060	Cell culture
Anti-PLIN2 (ADRP) antibody	Proteintech	15294-1-AP	Lipid droplet marker
Anti-PLIN3 (TIP47) antibody	Proteintech	10694-1-AP	Lipid droplet marker
Anti-α-tubulin antibody	Sigma-Aldrich	T6199	Mitotic spindle marker
BODIPY 493/503	Invitrogen	D3922	Neutral lipid staining
Bottle-top filter (0.22 μm)	NEST	WNB343001	Sterile filtration of OA–BSA solution
Bovine serum albumin (BSA)	Sigma-Aldrich	A8022	Blocking reagent
Clear nail polish	Commercially available	Not applicable	Sealing coverslips
Confocal microscope	Olympus	FV1200	High-resolution imaging
DAPI	Dojindo	340-07971	Nuclear staining
D-PBS (–)	Wako	049-29793	Cell culture
Dulbecco’s Modified Eagle’s Medium (DMEM)	Wako	041-30081	Cell culture medium
Ethanol (99.5%)	Wako	057-00456	Solvent for oleic acid
Fatty acid–free BSA	Sigma-Aldrich	A7030	Carrier protein for oleic acid
Fetal Bovine Serum	Gibco	10270-106	Medium supplement
Fluorescent secondary antibodies (Alexa Fluor–conjugated)	Commercially available	Not applicable	Species-specific secondary antibodies for immunofluorescence
FV10-ASW software	Olympus	Not applicable	Image acquisition and processing
Glass coverslips (φ15 mm)	Matsunami	2-176-03	Cell seeding for microscopy
Glass microscope slides	Matsunami	SFF-001	Glass slides for mounting coverslips
Glutaraldehyde (25% solution)	Wako	073-00536	Alternative fixation for PLIN3
HuH7 cells	JCRB Cell Bank	JCRB0403	Human hepatoma cell line
LipidTOX Red Neutral Lipid Stain	Invitrogen	H34476	Alternative lipid droplet stain
Nitrogen gas (N2)	Commercially available	Not applicable	Evaporation of ethanol
Oleic acid	Sigma-Aldrich	O1383	For preparation of oleic acid–BSA conjugates
Paraformaldehyde, 4% in PBS	Nacalai Tesque	09154-85	Fixation
Penicillin–Streptomycin	Gibco	15140-122	Antibiotics
Phosphate-buffered saline (×10)	Not applicable	Not applicable	Can be substituted with any vendor
RO-3306	Sigma-Aldrich	SML0569	CDK1 inhibitor for G2/M synchronization
SlowFade Diamond Antifade Mountant	Invitrogen	S36963	Mounting medium
Sodium chloride (NaCl)	Wako	191-01665	Preparation of 150 mM NaCl
Sodium hydroxide (NaOH)	Wako	080-01061	Used for saponification of oleic acid
Tali Cellular Analysis Slides	Thermo	T10794	Used for cell cycle analysis
Tali Image-based Cytometer	Thermo	Not applicable	Used for cell cycle analysis
Triton X-100	Sigma-Aldrich	T8787	Cell permeabilization
Widefield fluorescence microscope	KEYENCE	BZ-9000	Rapid screening of mitotic cells