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104 lines
3.5 KiB
Markdown
104 lines
3.5 KiB
Markdown
---
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name: Oil Red O Staining Protocol for Adherent Cells
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id: PROT-0015
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version: 1.0
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description: Protocol for staining and quantifying lipid droplets in adherent hepatocyte-like cells
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author: J. Jordan
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created: 2025-02-09
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materials:
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- Oil Red O powder
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- 100% isopropanol (2-propanol)
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- 4% paraformaldehyde (PFA)
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- PBS
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- Distilled water
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- 0.2-micron syringe filter
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- 96-well plate
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- Echo Revolution inverted microscope
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- Spectrophotometer (492 nm)
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steps:
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- "Prepare ORO staining solution"
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- "Fix cells with 4% PFA"
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- "Stain cells with filtered ORO solution"
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- "Wash and image cells"
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- "Extract ORO for quantification"
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- "Measure absorbance at 492 nm"
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notes: |
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Optimized for hepatocyte-like cells (HepG2, Huh7, AML12)
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Filtration of ORO solution is critical for good results
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Volume specifications are for 96-well plates - adjust for other formats
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---
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#Protocol for Oil red O (ORO) staining in adherent “hepatocyte-like” cells (e.g. HepG2, Huh7, AML12?)
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Adapted by J. Jordan; revised 02-09-25 by JJ.
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Note: Volumes are for 96-well plates. Adjust for larger well formats.
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1. Preparation of ORO staining solution:
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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”)
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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.
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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!
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5. Staining cells with ORO staining solution:
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6. Aspirate cell media and then wash twice with PBS.
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7. Add 75 ul cold 4% paraformaldehyde (PFA) to each well and allow fixation to occur for 20-30 m at room temperature (RT).
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8. Aspirate PFA.
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9. Wash cells twice with 100 ul PBS and aspirate to last wash until cells are very dry.
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10. Add 75 ul freshly prepared ORO solution to each well and stain for 30 m at RT.
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11. Wash twice with 150 ul distilled water.
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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.
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13. View and image cells in brightfield on Echo Revolution inverted microscope:
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14. Clip plate into stage.
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15. Adjust height of objectives with puck until cells are visible.
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16. Create a new folder to contain your images.
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17. Identify imaging parameters that will work for all of your wells so that images can be compared.
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18. Using identical imaging conditions (except for minor adjustments to focus), image all wells.
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19. Ensure images are indexed with their sample names and experimental methods.
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20. Transfer images to your Teams lab notebook data folder (USB or Airdrop) with image index.
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21. Extraction of ORO
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22. Add 75 ul of 100% isopropanol to each well and agitate for 5 minutes to extract ORO from cells.
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23. Transfer 60 ul isopropanol extraction to 96-well assay plate
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24. Add 60 ul pure isopropanol to at least 3 wells to account for background.
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25. Make sure plate reader is set to 492-nm protocol.
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26. Clip plate into spectrophotometer ensuring A1 is in the bottom-left corner.
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27. Label your data file with your experiment ID (usually your initials and the date you started your cell plate).
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28. Measure 492-nm absorbance.
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29. Export data onto a USB. |