We have a better working protocol to differentiate 3T3L1 cells. After reaching confluence induction starts DMEM high glucose+10% FBS+ 2uM rosiglitazone+ 250nM Dex+ 500uM IBMX +100nM insulin for 48 hrs, followed by DMEM high glucose+10% FBS+ 2uM rosiglitazone+ 100nM insulin for another 48 hrs, then into DMEM high glucose+10% FBS alone. Change every 48 hrs. Probably one or two  changes might be required, that means by 8 th day the cells will be completely differentiated. Almost 100 % with this protocol. 

I have seen the yellow color change in medium for 3T3L1 cells and the peeling off when I used to culture the cells in India. Probably the source it comes from often contain mycoplasma contamination. But if the cells are differentiating and if the differentiation has started in the correct density it should not occur. a slight change in color can be observed but the cells should survive. but if its too yellowish it can effect the experiments. 

 "The maturation medium containg (DMEM high glucose+FBS 10%+ insulin 10µg/ml) is very acidic."

How acidic, have you measured the pH?

As you probably know well that Phenol Red changes color from red-orange to orange-yellowish with only about a change in pH of about 0.2-0.3 pH units around pH 7.

Although I cannot speak of 3T3 L2 differentiation, many cells tolerate 0.2 units of pH change. Usually depletion of nutrients is not an issue in 24hrs but if you are concerned, measure glucose concentration in the medium. You can also add HEPES buffer, assuming it does not alter temporal changes for differentiation.

On the other hand, metabolic stress may be a component of triggering differentiation in which case, you wouldn't want to change a well-established protocol. 

Best, 

Dear ma'am,

You have mentioned here that 3T3-L1 differentiation medium is looking very acidic which should not be acidic because pH of differentiation medium should fall between 7.0 and 7.4 (Source: ATCC) So try to adjust media pH before use. Rest changing medium after 48 h is fine but try to add or remove media very gently just to avoid cell detachment as cells easily detach when differentiation occurs. 

I hope it may be helpful.

Regards,

thank you for your inputs.  Medium turning yellow withing 24h though HEPES is present in the media (pH around 7.3).  So, I replaced 50% of culture medium with fresh maturation medium.  Is there any hint how the cells would look etc to track the differentiation? oil red -O staining is end stage, i would like to know some thing in between.

regards,

Asavari

Kindly have a look at following articles, may be useful.

http://ajpgi.physiology.org/content/304/4/G420

http://pubs.rsc.org/en/content/articlehtml/2013/lc/c3lc50453k

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000300009

We routinely differentiate 3T3 cells into adipocytes and follow the every 2-day feeding protocol. The media does become yellow during this procedure (metabolically active cells secreting various factors) but in our hands as no real impact on differentiation. So I would not be too concern by this, at least for differentiating adipocytes.

Thanks for the inputs.  @ Satish, what are the changes in morphology when cells are differentiated?  In my case, the entire monolayer is now visible with naked eyes (like we see during trypsinization) and has started peeling off.  Would you mind sharing the protocol? esp upto which passage number generally cells are used for such expt? surface coating? etc

regards,

Asavari

Sure. Happy to help. 3T3 cells differentiate into adipocytes in a progressive manner taking 10-12days to become fully mature. Usually, you will start to see cells containing lipid droplets from Day 4 of differentiation onwards. Passage number of cell: This depends on how well you look after the cells at the preadipocyte stage. We keep them less than 50-60% confluent and have had cells differentiate well to passage 30. We don't normally use any coating though it might help in your case if the cells are floating off. Poly D-lysine is good for this and does not affect differentiation. Hope this helps and there are a number of good protocols out there which you can look at.

We have a better working protocol to differentiate 3T3L1 cells. After reaching confluence induction starts DMEM high glucose+10% FBS+ 2uM rosiglitazone+ 250nM Dex+ 500uM IBMX +100nM insulin for 48 hrs, followed by DMEM high glucose+10% FBS+ 2uM rosiglitazone+ 100nM insulin for another 48 hrs, then into DMEM high glucose+10% FBS alone. Change every 48 hrs. Probably one or two  changes might be required, that means by 8 th day the cells will be completely differentiated. Almost 100 % with this protocol. 

I have seen the yellow color change in medium for 3T3L1 cells and the peeling off when I used to culture the cells in India. Probably the source it comes from often contain mycoplasma contamination. But if the cells are differentiating and if the differentiation has started in the correct density it should not occur. a slight change in color can be observed but the cells should survive. but if its too yellowish it can effect the experiments. 

Thanks Nidhina.  In my hands cells are peeling off by day 6-7 and colour of the medium is yellow, not yellowish.  Cells for differentiation come from the 70-75% confluent flask.  I am thinking of  coating the plates with collagen or gelatin (available in lab, though poly-D-Lysine is suggested by Satish).  I will try rosiglitazone also.

Rosi, will speed up the process of differentiation and hence will mean that you could harvest at an earlier time-point (prior to the peeling off problem). However caution should be taken when using Rosi if you are interpreting any results that measure/quantify adipogenesis as an endpoint. If you simply want to differentiate cells into adipocytes then using Rosi is fine.

For peeling off issue, have you check that you incubators are correctly humidified??

 Yes I agree with Sathish, check the humidity of the chambers. If everything is proper this should not occur. And about changing the medium you can change it every 24 hrs if the medium is turning to yellow. It should not effect the cells. Also check the water source. Are you preparing the medium yourselves or buying from some commercial source??. 

Hello everyone,

thanks for the inputs.

The acidic medium problem is solved after shifting to Gibco FBS.  How? I dont know.  But I am facing new problem.  The cells are not peeling off.  After induction, cells should be more rounded, but mine are more fibroblastic and lot of debris appearing surrounding  the cells.  At D7 of differentiation, there is no significant lipid accumulation.  I am worried about the the debris as I feel, cells are disintegrating after putting them in maturation medium (DMEM, high glucose with pyruvate+10%FBS+Insulin).  Insulin is of bovine origin.  Whether this will make cells lyse?  Medium we are using ready made.

Please check for mycoplasma contamination. Its quite strange that cells disintegrate. can this debris be mycoplasma ??

Hi, I am culturing white and brown adipocytes. I can see every cell has droplets in it under microscope, but the medium did not turn yellow, it was still pink. Did the adipocytes differentiate successfully? Does medium have to turn yellow after differentiation? Thanks!