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

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.

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

Materials

Name	Company	Catalog Number	Comments
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