In this video, we demonstrate in vitro staining of neutral lipids in hepatocytes to detect hepatic steatosis or excessive fat accumulation in liver. Excessive fat is accumulated in the cells in form of lipid droplets. Oil Red O is a lipid soluble dye that binds to intracellular neutral lipids in these lipid droplets. When observed under a light microscope, these intracellular lipid droplets appear intensely red in color.
Protocol
1. Lipid staining with Oil-Red O Put a cell culture coverslip in every well in a 24-well plate. Seed 100,000 HepG2 cells per well. Add 1 mL of standard RPMI 1640. Pre-incubate at 37 °C and 5% CO2 for 24 h. Change standard RPMI 1640 medium for the steatogenic medium. Incubate for 24 h, 2 days, 3 days, and 4 days, refreshing the steatogenic medium every 24 h. After the appropriate time, discard the supernatant. Wash with 1 mL of p…
Disclosures
The authors have nothing to disclose.
Materials
Culture media RPMI 1640
ThermoFisher-Gibco, US
31800-022
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Fetal Bovine Serum (FBS)
ThermoFisher-Gibco, US
A4766801
–
HepG2 cell line
ATCC, US
HB-8065
Hepatocellular carcinoma human cells.
Humidified incubator
Thermo Electronic Corporation,US
Model: 3110
Temperature (37 °C ± 1 °C), humidity (90% ± 5%) , CO2 (5% ± 1%)
Inverted microscope Eclipse
NIKON, JPN
Model: TE2000-S
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Isopropanol
Sigma-Aldrich, US
I9030-4L
–
Oil Red O Kit
Abcam, US
ab150678
Kit for histological visualization of neutral fat.
Oil Red O Staining: A Technique for Staining Neutral Lipids in Hepatocytes for Detecting Hepatic Steatosis In Vitro. J. Vis. Exp. (Pending Publication), e21031, doi: (2023).