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
- 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 phosphate buffered saline (PBS). Discard the supernatant.
- Fix with 1 mL of 4% paraformaldehyde in PBS.
- Incubate for 1 h at room temperature.
- Discard the excess of paraformaldehyde.
- Rinse the cells with 1 mL of distilled water.
- Add 1 mL of 70% isopropanol and incubate for 5 min.
- Discard the excess of isopropanol. A PBS wash is not needed at this point.
- Add 1 mL of Oil-red O solution and incubate for 30 min.
- Discard the excess of the Oil-red O solution.
- Rinse with 1 mL of distilled water.
- Add 500 µL of hematoxylin solution. Incubate for 3 min.
- Discard the excess of hematoxylin solution.
- Rinse with 1 mL of distilled water.
- 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).