Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Summary

The present protocol describes how to use oil red O to dye lipid droplets (LDs), calculate the size and number of LDs in a fatty acid-induced fatty hepatocyte model, and use BODIPY 493/503 to observe the process of small LDs fusing into large LDs by live cell imaging.

Abstract

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Introduction

Lipid droplet (LD) accumulation in hepatocytes is the typical characteristic of non-alcoholic fatty liver disease (NAFLD), which can progress to liver fibrosis and hepatocellular carcinoma. It has been found that the earliest manifestation of fatty liver disease is steatosis, characterized by LD accumulation in the cytoplasm of the hepatocyte¹. Liver steatosis is invariably associated with an increased number and/or expanded size of LDs². LDs are thought to be generated from the endoplasmic reticulum (ER), consisting of triglyceride (TG) as the core, and are surrounded by proteins and phospholipids³. As the subcellular organelle responsible for TG storage, LDs exhibit different features regarding their size, number, lipid composition, proteins, and interaction with other organelles, all of which affect cell energy homeostasis⁴. The TG level is positively correlated with the size of LDs, and a higher intracellular TG content could form larger LDs⁵. LDs increase in size through the local synthesis of TG, lipid incorporation in the ER, and the fusion of multiple LDs⁶. Cells (adipocytes, hepatocytes, etc.) that contain large LDs have a special mechanism to efficiently increase lipid storage by LD fusion. The dynamic changes of LDs reflect the different energy metabolism states of the cell. It is crucial to develop methodologies that allow the observation and analysis of the various hepatic LDs in healthy and abnormal cells.

The main non-fluorescent dyes for LDs are Sudan Black B and oil red O. Sudan Black B stains neutral lipids, phospholipids, and steroids⁷. Oil red O is mainly used for staining LDs of skeletal muscle, cardiomyocytes, liver tissue, adipose cells, etc⁸., and is considered a standard tool for the quantitative detection of liver steatosis in mice and humans⁹. The dynamic change of LDs is mainly carried out by fluorescence dyeing. Nile red and BODIPY are both commonly used fluorescent lipid dyes^10,11. Compared with Nile red, BODIPY has stronger tissue permeability and binds better with LDs¹². BODIPY-labeled LDs can be used for staining living cells and colocalization with other organelles¹³.

The incidence of fatty liver disease is significantly higher in ruminant animals than in monogastric animals¹⁴. During the transition period, dairy cows experience a state of negative energy balance³. Large quantities of non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid, etc.) are synthesized into TGs in bovine hepatocytes, which leads to liver functional abnormality and greatly reduces the quality of milk products and production efficiency¹⁵. The present study aims to provide a protocol to analyze the size and the number of LDs, as well as to monitor the LD fusion dynamics. We constructed a model of LD formation by adding different concentrations of linoleic acid (LA) in hepatocytes¹⁶ and observed the changes in the size and the number of LDs during the process by staining LDs with oil red O. In addition, the process of the rapid fusion of LDs was also observed by staining with BODIPY 493/503.

Protocol

All procedures were approved and performed in accordance with the ethical standards of the Animal Care Committee of Henan Agricultural University (Henan Province, China).

1. Bovine hepatocyte cell culture

1. Thaw the primary hepatocyte cells¹⁷ and centrifuge 400 x g for 4 min at room temperature.
   NOTE: The primary hepatocyte cells were cultured and maintained following a previously published report¹⁷.
2. Discard the frozen storage solution with a pipette and suspend it with 1 mL of medium containing 10% fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM). Next, use a cell counting chamber to calculate the number of cells per milliliter, and then calculate the concentration of the above cell suspension and adjust the cell concentration to 1 x 10⁷ cells/mL.
   1. Add the cell suspension into a 60 mm cell culture dish containing 3 mL of the above medium. Culture the cells and incubate them at 37 °C and 5% CO₂ for 24 h.
3. When the cells grow to 80%, discard the medium and rinse with phosphate-buffered saline (PBS), then add 750 µL of 0.25% trypsin to digest the cells. Incubate the cells at 37 °C for 3 min, then neutralize them with the same amount of 10% FBS. Collect the cell suspension to centrifuge at 200 x g for 4 min at room temperature.

2. Oil red O staining

1. Add 800 µL of culture medium containing 10% FBS and DMEM into each well of the 24-well cell culture plate containing glass coverslips. Take 50 µL of the cell suspension (obtained in step 1.3) and add it to 950 µL of PBS to mix. Use a cell counting chamber to calculate the number of cells per milliliter, and then calculate the concentration of the above cell suspension. Finally, adjust the cell concentration to 4 x 10⁴/mL in each well and incubate at 37 °C and 5% CO₂ for 24 h.
2. After 24 h, discard the culture medium and wash with PBS. Suspend with 800 µL of DMEM induction medium containing 1 mg/mL bovine serum albumin (BSA).
3. Dissolve a total of 100 µL of LA (see Table of Materials) in 900 µL of anhydrous ethanol and prepare a standard solution (100 mmol/L). Add a gradient of 0 µmol/L, 100 µmol/L, 150 µmol/L, and 200 µmol/L LA into the 24-well plate. Repeat each treatment four times. Incubate at 37 °C and 5% CO₂ for 24 h.
4. Remove the culture medium and wash the cells with PBS three times. Fix with 400 µL of 4% paraformaldehyde for 20 min. Discard the fixative solution and wash it with PBS three times.
5. Incubate the cells with 60% isopropyl alcohol for 5 min, then discard it. Add freshly prepared oil red O working solution (see Table of Materials) (3:2 ratio of oil red O:water) for 20-30 min and discard the staining solution. Wash the cells with PBS two to five times until there is no excess dye solution.
6. Add 300 µL of hematoxylin staining solution (hematoxylin:water ratio of 1:10) and re-dye the nucleus for 1-2 min. Discard the dye solution and wash the cells with PBS two to five times. Take out the glass coverslips from the 24-well plate and place them on microscopic slides (the side with cells facing down) after dropping 10 µL of tablet sealant (see Table of Materials) onto the slide.
7. After sealing, observe and image the LDs of the cells under the oil lens of the optical microscope (see Table of Materials). Measure the diameter of the LDs by cellSens software and analyze the number and size of the LDs.

3. Measurement of the size and number of LDs

1. Capture images: Turn on the computer and microscope switch successively, place the slide on the loading platform, open the image analysis software (see Table of Materials), and connect the computer to view the image.
   1. Find the images at low power and drip an appropriate amount of cedar oil on the imaging slide. Adjust the observation factor to 100x, set automatic exposure, and capture the images by adjusting the appropriate field of view. Select three stained cell slides for each group for imaging.
2. Diameter measurement: Randomly select 60 LDs for each image to measure the diameters. After the measurement of each image, save the images and output the measurement results into a table for the subsequent analysis of the average size and distribution ratio of LDs.
3. Quantity measurement: Select three images for each stained and photographed slide, and randomly select three cells for quantity measurement in each picture. Count and analyze the number of LDs around the cell, and calculate the average number of LDs in the cell.

4. Dynamic observation of LD fusion

1. Follow the cell culture steps as mentioned in steps 1.1-1.3. When the cells grow to 80%, discard the medium and rinse with PBS, then digest them with 750 µL of trypsin for 3 min. Next, add 750 μL of the medium, centrifuge at 200 x g for 4 min at room temperature, and discard the supernatant.
2. Suspend the cells in 1 mL of culture medium, count, and adjust the cell concentration to 5 x 10⁵ cells/mL in a 35 mm dish. Culture at 37 °C and 5% CO₂ for 24 h.
3. When the cells have grown to 80%, change the medium to DMEM + 150 µmol/L LA for 24 h, and continue the culture in an incubator at 37 °C and 5% CO₂ to accumulate the LDs.
4. After 24 h, remove the culture medium. Wash the adherent cells with PBS and incubate them with 10 µg/mL BODIPY 493/503 neutral fluorescent probe (see Table of Materials) in the dark for 30 min. After incubation, wash the cells in the culture dish with PBS three times and add DMEM + 150 µmol/L LA.
   NOTE: Avoid light exposure during and after the 10 µg/mL BODIPY staining; 1 mg/mL BODIPY was diluted with PBS (1:100).
5. Place the culture dish in the groove of the microscope of the living cell station (see Table of Materials) to observe the dynamic changes of LDs. Turn on the power of the living cell workstation according to the starting sequence and avoid light.
   1. Turn on the power, transmission light source, microscope power source, mercury lamp fluorescent light source, charge-coupled device (CCD) camera power source, computer host power source, CO₂ valve, and CO₂ incubator.
6. Add distilled water to the groove on the loading platform, ensuring not to go over the air vent.
7. Turn on the computer and run "NIS-Elements". First, find the appropriate field of view on the 4x objective lens, then adjust it to the 40x objective lens successively. Select E100 for observing the sample in the microscope and L100 for previewing and photographing the sample on the computer.
   NOTE: E100 and L100 are microscope adjustment buttons on the living cell workstation (see Table of Materials), representing the eyepiece and the computer screen, respectively.
8. Design the parameters such as fluorescence channel (e.g., Ph-40x, Fluorescein isothiocyanate [FITC], shutter) and expected shooting time for the experiment. Set the shooting time in time, including the shooting interval time of 5 min between every two images and a total shooting time of 6 h.
   NOTE: Long-time shooting needs to use the perfect focus system (PFS) focus stabilization function. For this, first adjust the field of vision and the focus length, then click on PFS on the computer screen, and finally adjust the fine focus spiral.
9. Select different channel modes, single channel or all, select a different field of view, click preview, adjust the field of view, set these parameters, and click on start running to start shooting. Take a test shot for 5 min first; it can take a long time after the operation is normal.
10. After the shooting is performed, choose File > Save as > Save type > avi format to export the data to a video in 'avi' format. Use the '.nd2' image format to save the data program and export the photos.
11. For powering off the machine, turn it off in reverse order.

5. Statistics and result analysis

1. Analyze the data using one-way ANOVA. Report the results as the mean ± standard error.

Representative Results

The staining of cell LDs is shown in Figure 1. The red dots reflect cell LDs, and the blue dots reflect the nuclei. It can be seen that the size and number of LDs in each picture are different under the treatment of LA.

With the increase in LA dosage, the average diameter and number of LDs showed a significantly increasing trend, depending on LA concentration (Figure 2). As shown in Figure 2A, the number of LDs per cell was negatively correlated with different concentrations of LA. The median LD number per cell was 136 for the 100 µmol/L LA treated group, decreasing to 118 and 105 for the 150 µmol/L and 200 µmol/L LA treated groups, respectively. The average diameter of LDs in the control group was 0.72 µm, 1.38 µm for the 100 µmol/L LA treated group, 1.51 µm for 150 µmol/L LA treated group, and 1.64 µm for the 200 µmol/L LA treated group (Figure 2B).

In this study, LDs with a diameter <1 µm were defined as small, and LDs with a diameter >4 µm were defined as large. It was found that the distribution proportions of LDs also changed with the LA concentration, as shown in Figure 3; the proportion of small LDs decreased, and the proportion of large LDs increased. The proportion of small-diameter LDs was 43.33% at 100 µmol/L LA, 36.43% at 150 µmol/L LA, and 29.8% at 200 µmol/L LA. For large-diameter LDs, the highest proportion was 6.11% in 200 µmol/L LA, then 3.15% and 1.48% in 150 and 100 µmol/L LA, respectively. In the 200 µmol/L LA group, super large LDs (diameter >5 µm) were obviously observed, accounting for about 6.30%, higher than the 100 µmol/L LA (5.93%) and 150 µmol/L LA (4.82%) groups. The results indicated that a high concentration of LA increased the size of LDs and decreased the number of LDs. Meanwhile, the proportion of large LDs increased, and the proportion of small LDs decreased.

Large LDs were generally formed through the fusion of small LDs. LD fusion through live cell imaging was observed to prove this (Figure 4). Cells were treated with 150 µmol/L LA for 24 h and stained with BODIPY. Under the normal cell growing condition at 37 °C and 5% CO₂, the cells were observed continuously for 6 h with a live cell workstation. The images were captured every 5 min. As shown in Figure 4, in the first 15 min, there were still obvious smaller LDs. Then, the LDs slowly started to fuse from 20 min, and were completely fused into larger LDs by 35 min.

Figure 1: Lipid droplets. Hepatocyte cells cultured in different concentrations of LA and stained with oil red O. The red dots reflected LDs stained with oil red, and the blue-purple dots reflected the nucleus with hematoxylin. Three glass slides were selected for staining and imaging in each treatment. Scale bar = 10 µm. Please click here to view a larger version of this figure.

Figure 2: Number and size of LDs. Hepatocyte cells cultured in a concentration gradient from 0 to 200 µmol/L LA and stained with oil red O. (A) Average number of LDs per cell. The number of LDs in 27 cells was counted in each group. (B) The average diameter of LDs in a cell. The size of 60 LDs was counted in each group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Please click here to view a larger version of this figure.

Figure 3: LD proportion. Hepatocyte cells cultured in different concentrations of LA and stained with oil red O. Proportion ratio of LDs of different sizes were calculated. Please click here to view a larger version of this figure.

Figure 4: LD fusion. Hepatocyte cells were cultured with 150 µmol/L LA and incubated with the BODIPY 493/503 probe. After continuous observation for 6 h under a 40x magnification image, the LDs under the fluorescence and bright fields were photographed. The images were captured every 5 min. Fusion was marked with red boxes. Scale bar = 20 µm. Please click here to view a larger version of this figure.

Discussion

Depending on the pathological states, hepatic LDs undergo tremendous changes in their size and number. LDs are widely present in hepatocyte cells and play a key role in liver health and disease¹⁸. The quantity and size of LDs are the basis of the current research on the biogenesis of LDs¹⁹. The size and number of LDs for cells and tissues reflect their ability to store and release energy. The dynamic changes of LDs maintain the stability of lipid metabolic activities^20,21. An abnormal accumulation of LDs occurs in various pathological conditions and can be an indicator of metabolic disease^22,23. This protocol provides an accurate method to measure the size and quantity of LDs in a fatty acid-induced hepatocyte cell model and observe the fusion of LDs more efficiently and intuitively.

To study the size and dynamic changes of LDs is of great significance for understanding the occurrence of diseases related to lipid metabolism disorders and effective intervention. In this experiment, the key steps were the measurement and analysis of the size and quantity of LDs. First, different concentrations of LA were used to induce LD accumulation in hepatocytes. Other fatty acids, such as oleate and palmitate, could also be used to cause LD accumulation in hepatocyte cells^24,25. After the treatment with different concentrations of LA, it was found that the size of LDs was increased in a dose-dependent manner. These results indicated that the oil red O staining accurately measured the diameter of LDs. It was also found that a high concentration of LA could increase the proportion of large-sized LDs, suggesting that small LDs fused with each other rapidly. Rapid fusion is one of the dynamic changes in LDs; LD rapid fusion may occur within a few minutes or even tens of seconds, and it is mainly mediated by its surface phospholipids and protein^22,26. In the present study, LD fusion was clearly observed from 20 min to 35 min using BODIPY 493/503 labeling by a living cell workstation. The shooting interval between images can be set up in a few seconds to minutes, and the total shooting time can be set from 1 hour to several days, which provides convenience for observing the dynamic changes of LDs in different cells.

In terms of the staining analysis of the size of LDs, oil red O staining is more convenient and cheaper than fluorescent staining, and is more widely used to measure the apparent size of LDs⁹. It does not need to be treated against light. Moreover, the equipment requirements are not hard to achieve, and the oil lens of an ordinary optical microscope can be used for photo observation. Using this method, the researchers can select several groups of images to randomly measure the size and quantity of LDs; it is simple and convenient to operate, and can increase the sample size and reduce the error. Moreover, according to the measured and analyzed LD size, the distribution ratio of different LD sizes can be directly observed. Statistical analysis can be carried out by the above method after oil red O staining, and this method can also be applied to other cells, including a wide range of cells affected by lipid accumulation. In the early dyeing stage, it was found that oil red O was not easy to clean after dyeing, and there were substantial dye magazine residues. In the later stage, the dye was filtered, and the appropriate dyeing time was selected through continuous tests. After dyeing, increasing the cleaning times could avoid the above problem.

BODIPY 493/503 fluorescent dye is one of the most common probes for LD visualization²⁷. In this study, LD fusion was performed by labeling the LD green with BODIPY and observing the process with a living cell workstation. Nile red is also a commonly used fluorescent dye for neutral lipids, but its wide excitation band (450-560 nm), non-specific staining, poor tissue permeability, and other shortcomings may affect the results of LD staining to some extent. Compared with Nile red fluorescence staining, the excitation band of BODIPY 493/503 was narrower (460-490 nm), and it could be co-labeled with various fluorescent dyes¹¹. Secondly, BODIPY effectively avoids the interference of plant pigments²⁸. Finally, BODIPY has strong tissue permeability, low sensitivity to environmental polarity, and is not easy to quench¹²; therefore, it is widely used in fluorescence staining of LDs. However, the analysis of LD fusion has certain limitations. In order to obtain a continuous observation image of living cells, the lens cannot be moved, so only a local LD fusion can be observed in the vision, and the LD fusion efficiency of the whole cell cannot be analyzed more accurately.

In conclusion, this study provides a simple and reproducible method for LD morphology and size analysis, which can be widely applied to LD analysis in studying cellular lipid metabolism. This work also proved the process of LD fusion, laying the foundation for further study of LDs in the future.

Materials

0.25% trypsin	Gibco	25200072	reagent
4% paraformaldehyde	Solarbio	P1110	reagent
BODIPY 493/503	invitrogen	2295015	reagent
Cedar oil	Solarbio	C7140	reagent
cell counting chamber			equipment
cell culture dish	Corning	353002	material
cell sens software	Olympus IX73		software
Centrifuge	Eppendorf		equipment
DMEM	HyClone	SH30022.01	reagent
Fetal Bovine Serum	Gibco	2492319	reagent
hematoxylin	DingGuo	AR0712	reagent
Image view			image analysis sodtware
linoleic acid	Solarbio	SL8520	reagent
Live Cell Station	Nikon A1 HD25		equipment
NIS-Elements	Nikon		software
oil red O	Solarbio	G1260	reagent
optical microscope	Olympus IX73		equipment
Penicillin & Streptomycin 100×	NCM Biotech	CLOOC5	reagent
Phosphate Buffered Saline	HyClone	SH30258.01	reagent
Pipette	Eppendorf		equipment
Sealing agent	Solarbio	S2150	reagent