For those of you have experience in this field, I would like to ask you about this.
I performed 3T3-L1 for my research with usual method such as:
1. Seed the cell in 96 wells plate in proliferation medium (DMEM high glucose + 10%calf serum 1%penicilin-strptomicyn + Na pyruvat 1mM)
2. 2 days after 100% confluence (day 0), change the medium with differentiation medium (proliferation medium + 0.25 µM Dexamethason + 0.5mM IBMX + 10µg/ml Human Insulin (I2643 Sigma))
3. On day 2, change the medium with maintenance medium (Proliferation medium + 10µg/ml Human Insulin (I2643 Sigma)), and wait until the cells mature.
In my experience, I could see mature adipocyte beginning at day 6.
However, the problem is the mature cell distribution was not good. They were not spread well. Some spots have more mature adipocyte, others not. After Day 10, I usually only found +/- 40% mature cells per well (picture was attached). Only in the well treated by troglitazone, I could get 90% maturation per well.
Is this normal? What percent of mature cells per well do you normally get in your experience?
Is there something wrong in my protocol?
Thank you.
The protocol for differentiation seems right but we used FBS instead of calf.
In our experience the percentage of differentiation varies each time we get new cells. At present, we have cells obtained from ATCC some months ago, that differentiate almost 90-100% after 5 to 8 days but we also have others that differentiate 40-70%. When the percentage of differentiation is low, clusters of adipocytes are usually seen. In addition, we found that after some time (2-6 months), these cells decrease the ability to differentiate and we need to thaw new cells from liquid nitrogen. I hope this information will be useful for you.
We found that we can improve the percentage of differentiation if we keep the differentiation mixturen for 3 instead of 2 days. And differentiation is further improved with the presence of pioglitazone in the differentiation mixture. I hope this information will be useful.
I agree with Maria, you should switch to FBS when you are differentiating.
We worked with 3T3-L1 for years and found that the strain of cells was the best predictor for differentiation. The last strain we used was freshly bought from ATCC and showed the least percentage of diff. after 8 days.
We also found that the plastics we grew them on made a difference. For some reason they did not differentiate well in 24-wells from one company... In our hands Eppendorf plates worked best.
I hope this helps.
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