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^5,6,7,8. These organelles fragmented into small vesicles before being distributed to the two daughter cells, where they reassemble during the progression through the subsequent cell cycle³.

Lipid droplets (LDs) are neutral lipid–containing organelles composed of a core of neutral lipids surrounded by a phospholipid monolayer. They are associated with a diverse set of proteins and are widely found in many types of cells, including proliferating cells^9,10. The protein and lipid composition of LDs varies depending on cell and tissue type¹¹. Among these proteins, the perilipin (PLIN) family has been extensively studied, and PLIN2 stabilizes LDs in hepatocytes^12,13. In addition to structural stabilization, PLIN family proteins have been implicated in the regulation of LD dynamics and interactions with other cellular components¹⁴. LDs function not only as storage depots for triacylglycerol and cholesteryl ester but also as hubs for energy metabolism¹⁵, signaling pathways¹⁶, and stress adaptation¹⁷. In pathological conditions, such as metabolic dysfunction–associated steatohepatitis (MASH, previously referred to as NASH), LD accumulation is markedly increased compared with healthy control^18,19, highlighting its importance in disease biology. However, only limited observation of the spatial localization of LDs in dividing cells was available, despite extensive research on the dynamics of mitochondria, the endoplasmic reticulum, and the Golgi apparatus during mitosis. We analyzed LD localization during mitosis, using the method presented here. We have previously reported that LDs exhibited spatial distribution patterns distinct from those of other intracellular organelles²⁰.

Here, we provide a detailed protocol to investigate the distribution of LDs and other intracellular organelles during mitosis in hepatoma cells. The method includes cell cycle synchronization and fluorescent staining, enabling reproducible visualization of LD dynamics during mitosis. Representative results demonstrate that LDs display cell cycle–dependent changes in spatial distribution between interphase and mitosis, facilitating a comparative analysis of LD localization during cell division. This approach can be broadly applied to study organelle localization, dynamics, and metabolic regulation in both physiological and pathological contexts.

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 final volume to 1 L.

2. Preparation of oleic acid conjugated with BSA

1. Place 90 mg of oleic acid in a 50-mL beaker and add 2 mL of ethanol (99.5%) to dissolve it.
2. Add 100 µL of 5 N NaOH to the solution and stir until they are fully mixed.
3. Evaporate ethanol under a stream of nitrogen gas.
4. Add 10 mL of 150 mM NaCl and incubate the mixture at 60 °C for 5 min.
5. Add 12.5 mL of ice-chilled 24% (w/v) BSA stock solution slowly, in a dropwise manner, to the oleic acid solution (2.4) and gently stir the mixture continuously for 10 min to allow gradual and homogeneous mixing at room temperature.
6. Adjust the pH to 7.4 and bring the final volume to 25 mL with 150 mM NaCl. The final concentration of oleic acid will be 12.8 mM.
7. Filter the solution through a 0.22 µm filter under sterile conditions and store aliquots at 4 °C for up to 2 weeks or at -20 °C for longer storage.

3. G2/M boundary synchronization of cells

1. Prepare the necessary controls.
   1. Prepare untreated asynchronous cells without RO-3306 treatment as a baseline control.
   2. For oleic acid–treated conditions, prepare cells treated with oleic acid alone without synchronization as a baseline control.
   3. For fluorescence staining, perform single-staining controls for each fluorophore to verify signal specificity and exclude spectral overlap between channels (Supplementary Figure 1).
   4. Acquire images using sequential acquisition settings or appropriate filter configurations to minimize spectral overlap and channel crosstalk.
      ​NOTE: These controls are essential for distinguishing cell cycle–dependent changes from baseline organelle distribution and for validating fluorescence specificity.
2. Maintain HuH7 cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a 5% CO₂ incubator.
3. Place glass coverslips in a 35-mm culture dish. Seed 2 × 10⁵ cells per 35-mm dish in the medium described above and incubate the cells overnight at 37 °C in a 5% CO₂ incubator.
4. Wash cells twice with PBS and then incubate with fresh medium containing 9 µM RO-3306 and 0.5 mM oleic acid.
5. Incubate the cells for 20 h under standard culture conditions.

4. Removal of RO-3306 reagent from the synchronized cells to enter the mitotic phase

1. Wash cells (step 3.4) three to five times with PBS.
2. Wash cells once with fresh culture medium.
3. Incubate cells in fresh medium for 1 h at 37 °C in a 5% CO₂ incubator to allow entry into mitosis.
   NOTE: The removal of RO-3306 allows synchronized cells to enter the mitotic phase from the G2/M boundary. After washing out the reagent, a substantial proportion of cells enter mitotic phase within approximately 1 h, during which time, mitotic cells can be identified by cell rounding and chromosome condensation. The optimal timing to observe a specific phase of mitosis may vary depending on the cell type and culture conditions.

5. Sample preparation of mitotic cells

1. Fix cells with 4% paraformaldehyde in PBS for 10 min at room temperature.
   CAUTION: Mitotic cells tend to detach from the dish; avoid washing before fixation.
   NOTE: For PLIN3 staining, cells were fixed with a combination of 2% paraformaldehyde and 2% glutaraldehyde.
2. Permeabilize fixed cells with 0.1% (w/v) Triton X-100 in PBS for 10 min at room temperature.
3. Block cells with 1% (w/v) BSA in PBS for 1 h at room temperature.

6. Staining LDs and other cellular organelles

1. Carefully remove the glass coverslips from the culture dish using fine forceps and place them cell-side up in a humidified chamber to prevent drying.
   NOTE: Perform all subsequent staining steps in a humidified and light-protected environment to preserve fluorescence signals.
2. Wash cells twice with PBS and incubate with primary antibodies diluted in PBS for 1 h at room temperature.
3. Wash cells twice with PBS and incubate with secondary antibodies diluted in PBS for 1 h at room temperature.
4. Wash cells twice with PBS and stain with a neutral lipid fluorescent dye for 10 min at room temperature.
   NOTE: Dyes with distinct spectral properties were selected to enable flexible multicolor imaging and minimize spectral overlap with other organelle markers.
5. Wash cells twice with PBS and stain nuclei with a DNA-binding fluorescent dye (DAPI: excitation, 350–410 nm; emission, 420–450 nm) diluted 1:1,000 in PBS for 5 min.
6. Wash cells twice with PBS, then mount the coverslips on glass slides using an antifade mounting medium to preserve fluorescence during imaging.
7. Seal the edges of the coverslips with clear nail polish to prevent drying and movement during imaging.
   NOTE: Since fluorescent signals gradually fade, acquire images within 1 week after staining.

7. Observation of intracellular organelles and their subcellular localization

1. Acquire fluorescence images using widefield fluorescence microscopy to rapidly screen multiple cells and select representative interphase and mitotic cells, using low excitation intensity and rapid acquisition settings to minimize photobleaching.
2. Acquire images of the selected cells at appropriate magnification for fluorescence staining analysis.
   NOTE: Interphase cells typically exhibit an intact single nucleus positioned centrally in the cytoplasm. In contrast, mitotic cells display condensed chromosomes aligned in the equatorial plane of the bipolar spindle. These morphological features can serve as visual criteria for distinguishing interphase and mitotic cells.
3. Visualize LDs and nuclear morphology using appropriate filter sets for neutral lipid fluorescent dyes and a DNA-binding fluorescent dye by the widefield fluorescence microscopy.
   NOTE: Select filter sets according to the specifications of the microscope system used.
4. For higher-resolution validation, acquire fluorescence images using confocal microscopy equipped with a high-numerical aperture objective lens (e.g., 60×, NA ≥1.2). Detect fluorescence signals for neutral lipid fluorescent dyes and a DNA-binding fluorescent dye using appropriate excitation lasers and emission filter settings. Process images using commonly available image acquisition and analysis software.
   NOTE: Depending on the microscope system, either water- or oil-immersion objective lenses may be used. The choice of immersion medium should follow the manufacturer's recommendations and the experimental purpose. Widefield fluorescence microscopy is suitable for routine observation and comparison of LD distribution between interphase and mitotic cells, whereas confocal microscopy is recommended when a higher spatial resolution is required.

8. Quantification of the number and size of LDs

1. Acquire fluorescence images of cells under identical imaging settings for all conditions.
2. Open the acquired images using image analysis software.
3. Set appropriate thresholds to identify LDs and perform particle analysis to quantify lipid droplet number and area.
   NOTE: Imaging parameters should be kept constant across all conditions to ensure accurate comparison of LD number and area.

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