Does anyone can explain and perhaps give guidelines on how to read the lipid droplets or accumulation in 3T3-L1 cells after Oil Red Oil staining? This is because I cannot confidently calculated the percentage of lipid droplets or accumulation in 3T3-l1 cells. How we can determine it was 20%, 80% reduced or else.
Thank You
You can make morphometric measurements by determining the area that is positive for the stain. To increase accuracy, it will be best to photograph multiple fields and average your findings from each condition of tests.
If you have a decent image analysis software, it will make the process easier. All you will need to do is to set the threshold for what level stain is to be considered positive. Define the scale, if the software does not sense the magnification of the objective lens, and the rest is easy.
In the absence of such a software, you could use NIH imageJ, which is free. You will need to calibrate the scale manually if you want to find the actual area. For relative numbers, if you only want a % change, this calibration is not necessary.
All such measurements should be considered semi-quantitative.
After taking picktures remove all water and let dry and elute Oil Red O by adding 100% isopropanol, incubate about 10 min (can be longer)
Pipet the isopropanol with Oil Red O up and down several times to be sure that all Oil Red O is in the solution, Transfer to 1.5 ml tubes and Measure OD at 500 nm, 0.5 sec reading. As blank use 100% isopropanol.
From OD you can calculate % change in treatment and control groups.
Gudluck!
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining