oil red o staining for visualizing intracellular lipid droplet accumulation in hepg2 cells

Oleic Acid-Induced HepG2 Cells for Metabolic Disease Analysis 

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a range of conditions, including simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma1,2,3,4,5,6, all attributed to factors other than alcohol consuption7. MASLD is the most prevalent liver disease caused by metabolic liver injury, affecting nearly one-quarter of the global population8,9,10,11,12. While the precise pathogenesis of MASLD has not yet been elucidated, various theories attempt to explain its development. One prevailing notion suggests a departure from the classic &#34;two-hit&#34; theory towards a &#34;multiple-hit&#34; model1. Central to these hypotheses is the role of insulin resistance, which is believed to be pivotal in MASLD pathogenesis13. Research indicates that insulin resistance in hepatocytes leads to increased levels of free fatty acids, subsequently forming triglycerides stored within the liver14,15.
Researchers have used both in vivo and in vitro models to simulate fat deposition in MASLD; yet fully replicating its pathomechanism remains challenging. Despite this limitation, these models have been instrumental in studying potential therapeutic targets for MASLD. However, the development of a stable model of MASLD is crucial. While animal models are effective, they are time-consuming and expensive, thus highlighting the growing interest in in vitro cellular models. These models often use single or multiple free fatty acids such as oleic acid (OA) and palmitic acid to recreate diet-induced MASLD. Among these, the human hepatoblastoma cell line HepG2 is often used to establish in vitro cellular models of MASLD.
OA induction stimulates HepG2 cells to replicate fatty deposition akin to MASLD, a method with a well-established history. The aim of this study was to demonstrate the viability, Oil Red O (ORO) staining, lipid content, and triglyceride (TG) level of HepG2 cells treated with 0.25 mM OA. The objective of this experiment was to provide further evidence for the development of MAFLD modeling studies.

Author Spotlight: Establishing MASLD Cell Models for Investigating Disease Mechanisms and the Lipid-Lowering Effects of Koumiss

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

Our research direction is the study of traditional Mongolian medicine by establishing a metabolic dysfunction-associated steatotic liver disease cell model. The role and the mechanism of koumiss therapy are studied. The findings of the presence study will prove advantageous in the future, enabling the establishment of metabolic dysfunction-associated steatotic liver disease cell models and the investigation of the disease underlying mechanisms.A future study will be conducted based on the metabolic-dysfunction associated steatotic liver disease cell model to investigate the mechanism of action of the lipid-lowering effect of koumiss.

Oil Red O Staining for Visualizing Intracellular Lipid Droplet Accumulation in HepG2 Cells

To begin seed overnight grown culture of Hep G2 in DMEM in a six well plate. Culture the cells at 37 degrees Celsius for 24 hours. After incubation, add two milliliters of cell culture medium containing oleic acid to each well.Incubate the cells for 24 hours at 37 degrees Celsius. The next day, remove the culture medium and wash the cells twice with PBS. Add one milliliter of oil red O fixative to each well and incubate for 30 minutes.Discard the fixative and wash the cells twice with distilled water. Add one milliliter of 60%isopropanol to each well and incubate for 30 seconds. After discarding isopropanol, add one milliliter of freshly prepared oil red O stain solution and incubate for 20 minutes.Now, add one milliliter of 60%isopropanol and incubate for 30 seconds. Wash the cells five times with water to remove excess dye. After microscopic imaging, observe the cells covered with distilled water on a computer.After imaging, remove the water from the plate and allow it to dry. Add two milliliters of isopropanol to each well and shake the plate on an orbital shaker for 10 minutes. Transfer 100 microliters of cell suspension to a 96 well plate with 16 wells in each group.On a microplate reader at 510 nanometers, measure the optical density to calculate the lipid content. Compared to the control cells, Hep G2 cells treated with oleic acid showed increased red staining intensity, indicating higher lipid droplet formation. The lipid droplets and lipids in Hep G2 cells increased with rising oleic acid concentrations.

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Research

JoVE Journal

Biology

84 Views. To begin seed overnight grown culture of Hep G2 in DMEM in a six well plate. Culture the cells at 37 degrees Celsius for 24 hours. After incubation, add two milliliters of cell culture medium containing oleic acid to each well.Incubate the cells for 24 hours at 37 degrees Celsius. The next day, remove the culture medium and wash the cells twice with PBS. Add one milliliter of oil red O fixative to each well and incubate for 30 minutes.Discard the fixative and wash the cells twic...

Video: Oil Red O Staining for Visualizing Intracellular Lipid Droplet Accumulation in HepG2 Cells

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading to significant health challenges. Previous reports have studied the establishment of animal and cellular models for MASLD, highlighting differences between them. In this study, a cellular model was created by inducing fat accumulation in MASLD. HepG2 cells were stimulated with the unsaturated fatty acid oleic acid at various concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) to emulate MASLD. The model&#39;s efficacy was assessed using cell counting kit-8 assays, Oil Red O staining, and lipid content analysis. This study aimed to create a simple-to-operate cellular model for MASLD cells. Results from the cell counting kit-8 assays showed that the survival of HepG2 cells was dependent on the concentration of oleic acid, with a GI50 of 1.875 mM. Cell viability in the 0.5 mM and 1 mM groups were significantly lower than those in the control group (P &#60; 0.05). Furthermore, Oil Red O staining and lipid content analysis examined fat deposition at varying oleic acid concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) on HepG2 cells. The lipid content of the 0.25 mM, 0.5 mM, and 1 mM groups was significantly higher than that of the control group (P &#60; 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P &#60; 0.05).

This article describes the use of oleic acid-induced HepG2 cells as a model for metabolic dysfunction-associated steatotic liver disease.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

Assessing the Effect of Oleic Acid on HepG2 Cell Viability Using Cell Counting Kit-8

To begin seed overnight-grown culture of HepG2 in 100 microliters of DMEM in each well of a 96-well plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day discard the supernatant and add 100 microliters of oleic acid per well according to the specified grouping.For the control group, add 100 microliters of DMEM to each well. Incubate the cells at 37 degrees Celsius for 24 hours. After incubation, add 10 microliters of cell counting reagent to each well, mix gently, and incubate for two hours in the dark.Place the plate in the microplate reader and measure the absorbance at 450 nanometers. Calculate the GI50 values of HepG2 cells according to the optical density. Compared to the control, oleic acid decreased HepG2 cell survival rates at concentrations of 0.125, 0.25, 0.5, and one millimolar, with statistical significance observed at 0.5 and one millimolar.

Effect of Oleic Acid Concentrations on Total Triglyceride Levels in HepG2 Cell Supernatant

To begin, equilibrate the total triglyceride kit at room temperature for 20 minutes. Collect the supernatant of overnight grown Hep G2 cells and centrifuge at 1, 570 G for 10 minutes. Set the standard and test sample wells.Add 50 microliters of the oleic acid to the standard labeled wells. In addition to the blank and standard wells, add 10 microliters of cell culture to the sample wells. Then add 40 microliters of sample diluent to each well.Now add 100 microliters of detection antibody, horse radish peroxidase to each well. Seal with a plate membrane and incubate at 37 degrees Celsius in a constant temperature oven. After one hour, discard the supernatant and blot dry on dust-free paper.Add washing solution to each well and incubate at room temperature for one minute. Now, add 50 microliters of substrate A and 50 microliters of substrate B to each well. Mix gently and incubate for 15 minutes at 37 degrees Celsius.Add 50 microliters of termination solution, and within 15 minutes, measure the optical density at 450 nanometers. Plot the concentration of the standard along the x axis and the corresponding absorbance value along the Y axis. Perform linear regression and derive the curve equation to calculate the concentration value of each sample.Compared to the control group, treatment of Hep G2 cells with varying concentrations of oleic acid resulted in a significant increase in the total triglycerides content of the cell supernatant.