I m trying to differentiate 3T3-L1 adipocytes. Can anyone pls tell me, how many days it take for complete differentiation. I differentiated it for 8 days using standard protocol, but i didn't observe adequate differentiation, which was confirmed by Oil-red O staining. Any suggestion?
The cocktail including steroid and insulin is essential for the induction of adipocyte differentiation from 3T3-L1 cells. It takes about 5-7 days to express PPAR-gamma and AQP-7.
You should definitely observe lipid accumulation by 7 days (sometimes even earlier). Several parameters can have a large impact on differentiation efficiency, notably cell passage number, cell density (confluency required), and serum source. Please describe the composition of your differentiation medium, and the source of your L1a cells, how and in what type of vessel you cultured the cells, and this might offer some clue as to what went wrong. Also, make sure to check for lipid accumulation by phase microscopy, too (just to make sure you are not getting a false negative due to an unknown glitch with the Oil Red O staining procedure).
Thanks Dr Yoshida and Dr Cohen,
I used the following protocol:
Murine 3T3-L1 fibroblasts were obtained from cell repository of Natinal Centre for Cell Sciences, Pune, India. For differentiation into 3T3-L1 adipocytes, 3T3-L1 fibroblasts (passage no. 21-22) were seeded in 24-well plate and grown to confluence, after which the cells were cultured for 2 days in DMEM high glucose supplemented with 10% FBS, 1X Pen-Strep, 5μg/ml insulin, 0.5 mM 3- isobutyl-1-methylxanthine, 250nM dexamethasone (Sigma-Aldrich), followed by 6 days in DMEM high glucose containing 10% FBS, 1X Pen strep, and 5μg/ml insulin. Media was changed at every 2 days interval. After 8 days, Oil-red O staining was performed.
Dr Cohen, How would I check whether the cells have been differentiated by observing under phase contrast microscope?
Please also let me know, whether I can passage the adipocytes for further experiments?
You can see the lipid droplets as phase-bright spheres within the cells. Examples and many tips for culturing at ATCC's website: http://atcc.org/~/media/6124AF1E4C2A47CF904435117909AC25.ashx
As enumerated there, you want to hold your 3T3-L1s at confluence for 2 days prior to adding differentiation medium. Better differentiation is achieved at lower passages (you may be at too high a passage). There is a huge amount of lot-to-lot variability in FBS; not all support differentiation, and you may need to test several before finding a suitable lot. Try to get FBS that has been validated for L1s, and make sure you culture your stocks prior to differentiation in BCS not FBS. Also, try to make sure your stock plate of non-differentiated cells remain subconfluent while you are propagating them. I would not try to passage cells that were exposed to DMI.
You may also get some help from Dr. Gaikwad lab people from CDRI, they recently publised some data on this in Mol Cell Endocrinol. 2015 Jan 5;399:373-85. I appreciate all inputs from the eminent scientists in this form.
Passage number 22 is normally too high. Even you may observe some lipid droplets at the end of differentiation period, it is not even comparable with the differentiaton conducted with early passage cells. One additional issue is passaging protocol. You should never reach over %80 confluency during expansion. Otherwise, cells lose their differention capacity even if they are at early passages. If you overgrowth cells in any passage, you should get rid of them and buy a new batch. Therefore, if the cells are forced to differentiate by either DMI or overgrowing, passaging would not save your cells.
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