Differentiation protocol followed is standard: after 2 days of post confluence, differentiation is induced using dexa,Ibmx and insulin combination followed by addition of maintenance media every 2 days.
Oil red protocol followed is also generic: 0.5 g of oil red is dissolved in 100ml isopropanol by mixing it overnight then leaving it for 20min at 37C.This solution is then diluted in 3:2 ratio with water, incubated for 10 min at 37C then double filtered with 0.2 um syringe filter.
Staining protocol: After washing cells, they are fixed using formalin for 1 hour.Cells are then washed with water followed by incubation in 60 % isopropanol for 5 min.The diluted oil red stain is then added followed by incubation for 2 hours.Cells are checked for staining after 2-5 washes with water.
We use 0,3 g of oil-red/100 mL isopropanol.
How old is your oil-red stock? We keep it for max 1 year.
- You should filter the oil-red stock after it's been dissolved (and before dilution)
- Fixate you cells like you described.
- Make diluted oil-red just before use! 3:2 with water like you say, and incubate for 45 min to 1 h, then filter.
- Meanwhile you can wash you fixated cells with water and incubate with isopropanol
- we only incubate diluted oil-red for about 25 min
also:
- washing sides of wells with isopropanol reduces oil-red binding to the plastic surfaces
- some coating agents (like gelatin) may bind oil-red.
Good luck!
Thanks a lot for the tips...I had prepared the stock fresh and as you had mentioned always used a fresh dilution for staining . I have a few questions:
How long do you incubate cells with isopropanol after staining?What percentage of isopropanol do you use for washing sides of wells?
I will try your suggestions and see how it works.
Hi, I have never used oil red to look at 3t3l differentiatio, but I used Nile Red to look at lipid droplets. Are you sure your red clumps are not indeed big lipid droplets inside the cells? this is what you would expect after the differentiation. Do you observe the clumps also in non differentiated cells?
Hope this helps,
Good look,
Ingram
60% isopropanol for 5 minutes, just like you described. You wash the side of the wells with this solution as you add it to the wells. (alternatively, you may simply scale down the volumes so that the oil-red-O solution just covers the cells)
Ingram has a good point, I assume you're talking about unevenly shaped unspecific/unsoluble clumps of oil-red among smooth bubbles of lipids in the cells? You'll easily see the difference under the microscope. And yes, it's good to include non-diff control cells
Thank you....the clumps are like nonspecific bindings and doesnt appear like its bound to lipid droplets
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