Oil Red O Staining: A Technique for Staining Neutral Lipids in Hepatocytes for Detecting Hepatic Steatosis In Vitro

Published: April 30, 2023

Abstract

Source: Campos-Espinosa, A. et al., A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium. J. Vis. Exp. (2021)

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

  1. Put a cell culture coverslip in every well in a 24-well plate.
  2. Seed 100,000 HepG2 cells per well. Add 1 mL of standard RPMI 1640.
  3. Pre-incubate at 37 °C and 5% CO2 for 24 h.
  4. Change standard RPMI 1640 medium for the steatogenic medium.
  5. Incubate for 24 h, 2 days, 3 days, and 4 days, refreshing the steatogenic medium every 24 h.
  6. After the appropriate time, discard the supernatant.
  7. Wash with 1 mL of phosphate buffered saline (PBS). Discard the supernatant.
  8. Fix with 1 mL of 4% paraformaldehyde in PBS.
  9. Incubate for 1 h at room temperature.
  10. Discard the excess of paraformaldehyde.
  11. Rinse the cells with 1 mL of distilled water.
  12. Add 1 mL of 70% isopropanol and incubate for 5 min.
  13. Discard the excess of isopropanol. A PBS wash is not needed at this point.
  14. Add 1 mL of Oil-red O solution and incubate for 30 min.
  15. Discard the excess of the Oil-red O solution.
  16. Rinse with 1 mL of distilled water.
  17. Add 500 µL of hematoxylin solution. Incubate for 3 min.
  18. Discard the excess of hematoxylin solution.
  19. Rinse with 1 mL of distilled water.
  20. Observe under the microscope at a magnification of 400x (Objective 40x, Ocular 10x).

Divulgazioni

The authors have nothing to disclose.

Materials

Culture media RPMI 1640 ThermoFisher-Gibco, US 31800-022
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
Isopropanol Sigma-Aldrich, US I9030-4L
Oil Red O Kit Abcam, US ab150678 Kit for histological visualization of neutral fat.
Paraformaldehyde Sigma-Aldrich, US P6148-500G
Penicillin/streptomycin ThermoFisher-Gibco, US 15140-122 Antibiotics 10,000 U/mL Penicillin, 10,000 μg/mL Streptomycin
Phosphate buffered saline ThermoFisher-Gibco, US 10010-023
Serological Pipettes Sarstedt, AUS 86.1253.001 Non-pyrogenic, sterile, 5 mL
Serological Pipettes Sarstedt, AUS 86.1254.001 Non-pyrogenic, sterile, 10 mL
Sodium bicarbonate Sigma-Aldrich, US S5761-1KG Preparation of culture media
Sodium oleate Santa Cruz Biotechnology, US sc-215879A
Sodium palmitate Santa Cruz Biotechnology, US sc-215881
Trypsin 0.05% /EDTA 0.53 mM Corning, US 25-052-Cl
24 well cell culture cluster Corning, US 3524 Flat bottom with lid. Tissue culture treated. Nonpyrogenic, polystyrene, sterile. 1/Pack.

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Citazione di questo articolo
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).

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