I am trying to induce EMT in A549 cells by using TGF-beta ligand (sigma,T7039), I starve my cells for 24 hours in SFM and then add TGF-beta (5ng/ml) in SFM only and after 24 hours of this treatment, i make lysate of these cells. However after 24 hours of starvation I observe alot of vacuoles in my cells and dramatic change in morphology. There was no induction of EMT also, as there was no change in E-cadherin and vimentin levels (contro vs TGF-beta treated). I am attaching a picture of my cells after 24 hour of treatment with TGF-beta.
From this paper in Oncogenesis:
Cell culture and EMT induction
A549 was purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. To induce EMT, A549 cells were seeded at 70% confluence and cultured for 48 h in Dulbecco’s modified Eagle’s medium with 5% fetal bovine serum. TGF-β1 was added to a final concentration of 5 ng/ml.
http://www.nature.com/oncsis/journal/v2/n9/full/oncsis201337a.html
Looks likes serum starvation might not be required to induce EMT with TGF B1 in A549 cells. Anyway, it's worth a try. It sounds like the A549 cells don't tolerate serum starvation.
Try starving them in media with 2% serum, or charcoil-striped serum.
I would definitely suggest doing a range of serum reduced media (8%-0%). Some of my HNSCC lines don't show any changes in serum-free media, while others need some amount of serum just to survive. When my cell lines get sickly like that they are so focused on not dying that they don't waste extra energy undergoing simply cancer-related processes like EMT. I'd figure out how to keep the cells happy before attempting to induce any changes. A colleague recommends 5ng/mL TGF-beta for 48-72 hours works in his hands. They use low serum (.25%) starting 24 hours prior to TGF-beta treatment.
Thank you everyone. Steven can you tell me that the low serum condition you are suggesting it should be maintained during the treatment also?
I just ran across this, sorry it's so late. I use this cell model/treatment. Starvation will not affect the EMT too much. What is important is that the cells are not too confluent, start with a lower density. My typical protocol is to seed at 3000/cm2, starve the next day in 0.5% FBS and then the following day treat with 100 pM. Change the media the next day with 100 pM TGFβ. After 48 hrs, lyse for EMT analysis. Also, be sure to treat in 0.5% FBS media.
Adam can you please give me a reference of this dose i didnt come across it in the literature
i found some http://www.sciencedirect.com/science/article/pii/S0959804912009550
Hi Saba!
Where you able to induce EMT in A549 cells using tgf beta? What percentage of serum did you use? I am trying to using to induce EMT in A431 cells using EGF (serum free media). However, I am not getting consistent reduction in epithelial markers. Please let me know if I can change the conditions. Thanks
Yes, now its working. I am using 0.5% serum concentration in the media, in which I starve cells for 24hours prior to TGF beta treatment and then treat A549 cells for 48hours with 5ng/ml TGF-Beta in 0.5% FBS containing media only.
Hi, I am recently trying to induce A549 to EMT by TGF-beta-1, However, I can't see any change of morphology. I use serum free to starve the cell 24h and then treated the A549 cells with serum free medium with 5ng/mL tgf-b-1 for 48h.
Is there any problems for the serum free medium in this induction of EMT, or I should use low-serum medium？
5x10^5 per well at 6-well plate which is 50% confluency at seeding. then I starved the cells for 24h with serum-free medium they grew a little bit in starving, when I treated them with TGF-beta, they are ~65% confluency.
another thing, I used serum-free medium, is there any thing I should pay attention about serum-free, 1% serum or 5% serum. which condition you recommended.
Hi all, I am doing TGFB treatment i n EPH4 cells. I starve cells with 0.1% serum contained media for 3 h. Should I remove starvation media from cells and add fresh low serum media containing TGFB? or add a appropriately diluted TGFB to starvation media? Thanks
As long as your cells looks fine after starvation you don't need to do anything. But if they don't look good you may add serum from 0.5-5%. There are many papers which have used SFM as well as low serum conditions. It depends upon your cell line how sensitive they are towards serum withdrawal.
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