docs/Protocols/Oil red O in adherent cells protocol.md

---
name: Oil Red O Staining Protocol for Adherent Cells
id: PROT-0015
version: 1.0
description: Protocol for staining and quantifying lipid droplets in adherent hepatocyte-like cells
author: J. Jordan
created: 2025-02-09
materials:
  - Oil Red O powder
  - 100% isopropanol (2-propanol)
  - 4% paraformaldehyde (PFA)
  - PBS
  - Distilled water
  - 0.2-micron syringe filter
  - 96-well plate
  - Echo Revolution inverted microscope
  - Spectrophotometer (492 nm)
steps:
  - "Prepare ORO staining solution"
  - "Fix cells with 4% PFA"
  - "Stain cells with filtered ORO solution"
  - "Wash and image cells"
  - "Extract ORO for quantification"
  - "Measure absorbance at 492 nm"
notes: |
  Optimized for hepatocyte-like cells (HepG2, Huh7, AML12)
  Filtration of ORO solution is critical for good results
  Volume specifications are for 96-well plates - adjust for other formats
---

#Protocol for Oil red O (ORO) staining in adherent “hepatocyte-like” cells (e.g. HepG2, Huh7, AML12?)

Adapted by J. Jordan; revised 02-09-25 by JJ.

  

Note: Volumes are for 96-well plates. Adjust for larger well formats.

  

1. Preparation of ORO staining solution:
    

2. If necessary, prepare ORO stock solution by dissolving 0.175 g ORO powder (on the chemical shelf) in 50 ml 100% 2-propanol (aka “isopropanol”)
    
3. Dilute ORO stock solution in distilled water (dH2O) (i.e., Add 3 parts ORO solution to 2 parts dH2O) and vortex solution immediately before using to stain cells.
    
4. Add diluted ORO solution to a syringe with a 0.2-micron filter, then filter into a fresh vial. Skipping this filtration WILL ruin your experiment!
    

5. Staining cells with ORO staining solution:
    

6. Aspirate cell media and then wash twice with PBS.
    
7. Add 75 ul cold 4% paraformaldehyde (PFA) to each well and allow fixation to occur for 20-30 m at room temperature (RT).
    
8. Aspirate PFA.
    
9. Wash cells twice with 100 ul PBS and aspirate to last wash until cells are very dry.
    
10. Add 75 ul freshly prepared ORO solution to each well and stain for 30 m at RT.
    
11. Wash twice with 150 ul distilled water.
    
12. If imaging, add 100 ul of PBS to each well to improve microscopy. If skipping to extractions, you can leave the wells dry and proceed to Part IV.
    

13. View and image cells in brightfield on Echo Revolution inverted microscope:
    

14. Clip plate into stage.
    
15. Adjust height of objectives with puck until cells are visible.
    
16. Create a new folder to contain your images.
    
17. Identify imaging parameters that will work for all of your wells so that images can be compared.
    
18. Using identical imaging conditions (except for minor adjustments to focus), image all wells.
    
19. Ensure images are indexed with their sample names and experimental methods.
    
20. Transfer images to your Teams lab notebook data folder (USB or Airdrop) with image index.
    

21. Extraction of ORO
    

22. Add 75 ul of 100% isopropanol to each well and agitate for 5 minutes to extract ORO from cells.
    
23. Transfer 60 ul isopropanol extraction to 96-well assay plate 
    
24. Add 60 ul pure isopropanol to at least 3 wells to account for background.
    
25. Make sure plate reader is set to 492-nm protocol.
    
26. Clip plate into spectrophotometer ensuring A1 is in the bottom-left corner.
    
27. Label your data file with your experiment ID (usually your initials and the date you started your cell plate). 
    
28. Measure 492-nm absorbance.
    
29. Export data onto a USB.