Name: Oil Red O Staining and Measurement of Lipid Droplets (LDs) in Bovine Hepatocyte Cells
Uploaded: 2023-03-10T14:40:00
Description: Watch this Scientific Journal Video about Oil Red O Staining and Measurement of Lipid Droplets LDs in Bovine Hepatocyte Cells at JoVE.com

oil red o staining and measurement of lipid droplets (lds) in bovine hepatocyte cells

使用 Oil Red O 和 BODIPY 对脂滴大小和融合进行活细胞成像

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.

作者聚焦：牛肝细胞中脂滴大小和融合的评估  

牛肝细胞中脂滴大小和融合的评估

Our research focus on the lipid droplet number and the size induced by linoleic acid in liver cells. Additionally, we aim to provide insights into the mechanism of the fatty liver. Recent advancements in this field of research, suggests that choline, CCT alpha, established induced autophagy, can regulate lipid droplet size in bovine hepatic cells.Moreover, living cell workstation observations have shown that phospholipids have a considerable effect on lipid droplet fusion. Currently, gene aiding, fluorescence tweezer, liquid chromatography, omics analysis, single chromatography, flow cytometry, and other technologies are being used to group the vast research in this field. The experimental method has some limitations.For example, the phenomenon of limited fields can only be observed for lipid droplet fields and under a certain flight of waste. Oil Red O staining is more convenient, less expensive, and more widely used to measure the apparent size of lipid droplets than lipid droplet fluorescence staining. It is simple to operate and requires less equipment.Furthermore, we directly observe this phenomenal of lipid fusion through the living cell station. This study provide a simple and repeatable method to observe lipid droplet size and fusion, which can be widely applied to lipid droplet analysis in cellular lipid metabolism research. We will continue to conduct intensive research on lipid droplets, focusing on the mechanism of lipid droplet fusion and the structure changes of fatty liver in dairy cows.

Watch this Scientific Journal Video about Oil Red O Staining and Measurement of Lipid Droplets LDs in Bovine Hepatocyte Cells at JoVE.com

Oil Red O Staining and Measurement of Lipid Droplets (LDs) in Bovine Hepatocyte Cells  

油红O染色和牛肝细胞中脂滴（LDs）的测量

首先，取一个先前就座的24孔培养板，并在每个孔中放置一个玻璃盖玻片。然后加入含有10%FBS和DMEM的800微升培养基。调整细胞浓度后。在 37 摄氏度和 5% 二氧化碳下孵育 24 小时。然后丢弃培养基并用PBS洗涤细胞。将细胞悬浮在含有BSA的800微升DMEM诱导培养基中。将亚油酸溶解在无水乙醇中，制备浓度为每升100毫摩尔的标准溶液。在板中以递增的梯度加入亚油酸，重复四次。然后将板在 37 摄氏度和 5% 二氧化碳下孵育 24 小时。在孵育结束时，除去培养基并用PBS洗涤细胞三次。用 400 微升 4% 多聚氰醛固定 20 分钟。然后丢弃固定液并再次洗涤。用60%异丙醇孵育细胞五分钟，然后丢弃。加入新鲜制备的油红O工作溶液，20至30分钟后丢弃。用PBS洗涤细胞两到五次，直到去除多余的染料溶液。用300微升苏木精溶液重新染色细胞核一到两分钟。然后丢弃染料溶液并再次洗涤。从PBS洗涤板上取下玻璃盖玻片，并将其细胞面朝下放置在含有10微升片剂密封剂的显微镜载玻片上。要测量LD的大小和数量，请依次打开计算机和显微镜开关，并将载玻片放在装载平台上。然后打开图像分析软件。找到低功率的图像，并在成像载玻片上滴上适量的雪松油。逐渐调整物镜200倍并调整视野，直到找到清晰的图像。然后从所需区域捕获图像并保存。随机为每个图像选择 60 个 LD 并测量直径。将测量结果导出到表中，以分析LD的平均大小和分配比例。染色的肝细胞培养细胞显示出不同大小和数量的LD。在不同浓度下进行亚油酸处理。每个细胞的LDs数量与不同浓度的亚油酸呈负相关。每升100微摩尔亚油酸处理组每个细胞的中位LD数为136，并且在高浓度下降低。对照组LDs的平均直径为0.72 μm，随浓度的增加而增大。高亚油酸浓度增加了大LDs的比例，降低了小LDs的比例。

Research

JoVE Journal

Biology

Oil Red O Staining and Measurement of Lipid Droplets (LDs) in Bovine Hepatocyte Cells

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

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 ...