Name: A Fluorescence-Based Method To Visualize Lipid Droplet And Organelle Dynamics During Mitosis in Hepatoma Cells (Video) | JoVE Journal
Uploaded: 2026-05-15T14:30:00.000+00:00
Duration: 7 min 10 s
Description: Discover a fluorescence-based method to visualize lipid droplet dynamics during mitosis in hepatoma cells. This protocol enables researchers to study organelle redistribution and metabolic regulation in cancer cell biology.

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5 minutes

This study was supported by the Showa University Grant for Young Researchers and JSPS KAKENHI Grant Number 24K10084.

1. Stock solutions required for this protocol
<ol>
	<li>Prepare 24% (w/v) fatty acid&#8211;free bovine serum albumin (BSA) solution dissolved in 150 mM NaCl.</li>
	<li>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 NaH&#8322;PO&#8324;&#183;2H&#8322;O, 29 g of Na&#8322;HPO&#8324;&#183;12H&#8322;O, 80 g of NaCl, and 2.0 g of KCl in distilled water and adjusting the final volume to 1 L.</li>
</ol>
2. Preparation of oleic acid conjugated with BSA
<ol>
	<li>Place 90 mg of oleic acid in a 50-mL beaker and add 2 mL of ethanol (99.5%) to dissolve it.</li>
	<li>Add 100 &#181;L of 5 N NaOH to the solution and stir until they are fully mixed.</li>
	<li>Evaporate ethanol under a stream of nitrogen gas.</li>
	<li>Add 10 mL of 150 mM NaCl and incubate the mixture at 60 &#176;C for 5 min.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Filter the solution through a 0.22 &#181;m filter under sterile conditions and store aliquots at 4 &#176;C for up to 2 weeks or at -20 &#176;C for longer storage.</li>
</ol>
3. G2/M boundary synchronization of cells
<ol>
	<li>Prepare the necessary controls.
	<ol>
		<li>Prepare untreated asynchronous cells without RO-3306 treatment as a baseline control.</li>
		<li>For oleic acid&#8211;treated conditions, prepare cells treated with oleic acid alone without synchronization as a baseline control.</li>
		<li>For fluorescence staining, perform single-staining controls for each fluorophore to verify signal specificity and exclude spectral overlap between channels (Supplementary Figure 1).</li>
		<li>Acquire images using sequential acquisition settings or appropriate filter configurations to minimize spectral overlap and channel crosstalk. 
		&#8203;NOTE: These controls are essential for distinguishing cell cycle&#8211;dependent changes from baseline organelle distribution and for validating fluorescence specificity.</li>
	</ol>
	</li>
	<li>Maintain HuH7 cells in Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin at 37 &#176;C in a 5% CO&#8322; incubator.</li>
	<li>Place glass coverslips in a 35-mm culture dish. Seed 2 &#215; 105 cells per 35-mm dish in the medium described above and incubate the cells overnight at 37 &#176;C in a 5% CO&#8322; incubator.</li>
	<li>Wash cells twice with PBS and then incubate with fresh medium containing 9 &#181;M RO-3306 and 0.5 mM oleic acid.</li>
	<li>Incubate the cells for 20 h under standard culture conditions.</li>
</ol>
4. Removal of RO-3306 reagent from the synchronized cells to enter the mitotic phase
<ol>
	<li>Wash cells (step 3.4) three to five times with PBS.</li>
	<li>Wash cells once with fresh culture medium.</li>
	<li>Incubate cells in fresh medium for 1 h at 37 &#176;C in a 5% CO&#8322; 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.</li>
</ol>
5. Sample preparation of mitotic cells
<ol>
	<li>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.</li>
	<li>Permeabilize fixed cells with 0.1% (w/v) Triton X-100 in PBS for 10 min at room temperature.</li>
	<li>Block cells with 1% (w/v) BSA in PBS for 1 h at room temperature.</li>
</ol>
6. Staining LDs and other cellular organelles
<ol>
	<li>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.</li>
	<li>Wash cells twice with PBS and incubate with primary antibodies diluted in PBS for 1 h at room temperature.</li>
	<li>Wash cells twice with PBS and incubate with secondary antibodies diluted in PBS for 1 h at room temperature.</li>
	<li>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.</li>
	<li>Wash cells twice with PBS and stain nuclei with a DNA-binding fluorescent dye (DAPI: excitation, 350&#8211;410 nm; emission, 420&#8211;450 nm) diluted 1:1,000 in PBS for 5 min.</li>
	<li>Wash cells twice with PBS, then mount the coverslips on glass slides using an antifade mounting medium to preserve fluorescence during imaging.</li>
	<li>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.</li>
</ol>
7. Observation of intracellular organelles and their subcellular localization
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>For higher-resolution validation, acquire fluorescence images using confocal microscopy equipped with a high-numerical aperture objective lens (e.g., 60&#215;, NA &#8805;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&#39;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.</li>
</ol>
8. Quantification of the number and size of LDs 
<ol>
	<li>Acquire fluorescence images of cells under identical imaging settings for all conditions.</li>
	<li>Open the acquired images using image analysis software.</li>
	<li>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.</li>
</ol>

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</ol>

The authors declare that they have no conflicts of interest regarding the contents of this article.

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...

The cell cycle is a highly orchestrated event that ensures the accurate duplication and segregation of chromosomal DNA1,2, during which intracellular organelles are dynamically reorganized3. It consists of four major phases: G1, S, G2, and M1,2. 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 mitosis5,6,7,8. 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&#8211;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 cells9,10. 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 hepatocytes12,13. 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&#8211;associated steatohepatitis (MASH, previously referred to as NASH), LD accumulation is markedly increased compared with healthy control18,19, 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&#8211;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.

<table><tbody><tr><td>0.25% Trypsin/EDTA</td><td>Wako</td><td>201-16945</td><td>Cell dissociation</td></tr><tr><td>10 cm dish</td><td>Corning</td><td>430167</td><td>Cell culture</td></tr><tr><td>35 mm tissue culture dish</td><td>IWAKI</td><td>3000-035</td><td>Cell culture</td></tr><tr><td>6 cm tissue culture dish</td><td>IWAKI</td><td>3010-060</td><td>Cell culture</td></tr><tr><td>Anti-PLIN2 (ADRP) antibody</td><td>Proteintech</td><td>15294-1-AP</td><td>Lipid droplet marker</td></tr><tr><td>Anti-PLIN3 (TIP47) antibody</td><td>Proteintech</td><td>10694-1-AP</td><td>Lipid droplet marker</td></tr><tr><td>Anti-&amp;alpha;-tubulin antibody</td><td>Sigma-Aldrich</td><td>T6199</td><td>Mitotic spindle marker</td></tr><tr><td>BODIPY 493/503</td><td>Invitrogen</td><td>D3922</td><td>Neutral lipid staining</td></tr><tr><td>Bottle-top filter (0.22 &amp;mu;m)</td><td>NEST</td><td>WNB343001</td><td>Sterile filtration of OA&amp;ndash;BSA solution</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A8022</td><td>Blocking reagent</td></tr><tr><td>Clear nail polish</td><td>Commercially available</td><td>Not applicable</td><td>Sealing coverslips</td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1200</td><td>High-resolution imaging</td></tr><tr><td>DAPI</td><td>Dojindo</td><td>340-07971</td><td>Nuclear staining</td></tr><tr><td>D-PBS (&amp;ndash;)</td><td>Wako</td><td>049-29793</td><td>Cell culture</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Wako</td><td>041-30081</td><td>Cell culture medium</td></tr><tr><td>Ethanol (99.5%)</td><td>Wako</td><td>057-00456</td><td>Solvent for oleic acid</td></tr><tr><td>Fatty acid&amp;ndash;free BSA</td><td>Sigma-Aldrich</td><td>A7030</td><td>Carrier protein for oleic acid</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10270-106</td><td>Medium supplement</td></tr><tr><td>Fluorescent secondary antibodies (Alexa Fluor&amp;ndash;conjugated)</td><td>Commercially available</td><td>Not applicable</td><td>Species-specific secondary antibodies for immunofluorescence</td></tr><tr><td>FV10-ASW software</td><td>Olympus</td><td>Not applicable</td><td>Image acquisition and processing</td></tr><tr><td>Glass coverslips (&amp;phi;15 mm)</td><td>Matsunami</td><td>2-176-03</td><td>Cell seeding for microscopy</td></tr><tr><td>Glass microscope slides</td><td>Matsunami</td><td>SFF-001</td><td>Glass slides for mounting coverslips</td></tr><tr><td>Glutaraldehyde (25% solution)</td><td>Wako</td><td>073-00536</td><td>Alternative fixation for PLIN3</td></tr><tr><td>HuH7 cells</td><td>JCRB Cell Bank</td><td>JCRB0403</td><td>Human hepatoma cell line</td></tr><tr><td>LipidTOX Red Neutral Lipid Stain</td><td>Invitrogen</td><td>H34476</td><td>Alternative lipid droplet stain</td></tr><tr><td>Nitrogen gas (N&lt;sub&gt;2&lt;/sub&gt;)</td><td>Commercially available</td><td>Not applicable</td><td>Evaporation of ethanol</td></tr><tr><td>Oleic acid</td><td>Sigma-Aldrich</td><td>O1383</td><td>For preparation of oleic acid&amp;ndash;BSA conjugates</td></tr><tr><td>Paraformaldehyde, 4% in PBS</td><td>Nacalai Tesque</td><td>09154-85</td><td>Fixation</td></tr><tr><td>Penicillin&amp;ndash;Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Antibiotics</td></tr><tr><td>Phosphate-buffered saline (&amp;times;10)</td><td>Not applicable</td><td>Not applicable</td><td>Can be substituted with any vendor</td></tr><tr><td>RO-3306</td><td>Sigma-Aldrich</td><td>SML0569</td><td>CDK1 inhibitor for G2/M synchronization</td></tr><tr><td>SlowFade Diamond Antifade Mountant</td><td>Invitrogen</td><td>S36963</td><td>Mounting medium</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Wako</td><td>191-01665</td><td>Preparation of 150 mM NaCl</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Wako</td><td>080-01061</td><td>Used for saponification of oleic acid</td></tr><tr><td>Tali Cellular Analysis Slides</td><td>Thermo</td><td>T10794</td><td>Used for cell cycle analysis</td></tr><tr><td>Tali Image-based Cytometer</td><td>Thermo</td><td>Not applicable</td><td>Used for cell cycle analysis</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Cell permeabilization</td></tr><tr><td>Widefield fluorescence microscope</td><td>KEYENCE</td><td>BZ-9000</td><td>Rapid screening of mitotic cells</td></tr></tbody></table>

a fluorescence-based method to visualize lipid droplet and organelle dynamics during mitosis in hepatoma cells

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&#8211;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&#8211;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.

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 boundary20,21. 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 (<strong class="...

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

Intracellular lipid droplets (LDs) are ubiquitous in several cell types. LDs store neutral lipids such as triacylglycerol and cholesteryl ester and play ...

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Discover a fluorescence-based method to visualize lipid droplet dynamics during mitosis in hepatoma cells. This protocol enables researchers to study organelle redistribution and metabolic regulation in cancer cell biology.

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

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