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.
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.
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 dyes10,11. 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.
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
2. Oil red O staining
3. Measurement of the size and number of LDs
4. Dynamic observation of LD fusion
5. Statistics and result analysis
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.
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 activities20,21. An abnormal accumulation of LDs occurs in various pathological conditions and can be an indicator of metabolic disease22,23. 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 cells24,25. 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 protein22,26. 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.
The authors have nothing to disclose.
This research was jointly supported by the National Natural Science Foundation of China (U1904116).
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 |