We have grown HT-29 cells (serum free) on non-coated transwells (8 um pore size, Greiner Bio) and incubated these at the basolateral side with 2.5, 5 and 10 ng/ml TGF-beta 1 for 12-24-48-72 hrs. We measured cell migration by staining cells at bottom side of the transwell membrane with crystal violet according to standard protocols. Surprisingly we observed in all TGF-beta 1 conditions a reduced migration despite TGF-beta 1 is supposed to induce migration. However, we did observe a FCS-induced migration. Anybody any idea what might explain the TGF-beta 1 results? Thanks!
It's likely that your serum-free medium does not have enough basic ingradients for the cells to be growing normally and that may attribute to its poor response to TGFb1. On the other hand, FCS provides more than necessary goods for cells to respond (to something carried over in the FCS).
did you check the expression of tgfb receptor on these cells? Some cells do not express the receptor.
If your protocol matches previously published methods, e.g., Chen J Biol Chem. 287:17016, 2012, and your TGF-b1 has been shown to be active in some other bioassay, e.g. proliferation inhibition, check to be sure that your cells are really HT-29's.
Hi! Cell migration is also dependent of ECM proteins. In your serum free medium assays your cells may lacks ECM proteins to activate integrins. We generally coated both sides of the transwell filter with a solution of collagen I, collagen IV, laminin I or fibronectin, -10µg/ml in PBS (test which one is the best), ON/N at 4°C or 3h at 37°C). The cells will therefore be able to migrate on the ECM. Also, HT29 cell migration on transwell filter is highly dependent of cell density. Be sure to work with enough cells.
The FCS contains a lto of plasmatic ECM proteins (FN, vitronectin...) supporting cell migration.
We have used fibronectin to coat only the bottom side of the transwell. This seems to be enough.
I agree with you Lisette that's enought but, cells will then migrate toward a gradient of FN, a migration called "haptotaxism" as Johan want to performe chemotaxy assays I think that it is more relevent to coat both sides of the filter. Cells will be attract by the TGF only
Agreed with the comments regarding the medium. I work with HT29 cells as well and they are not very happy in serum-free conditions.
I have been doing the same experiment but with Panc-1 cells, and I observed inhibition with TGF-b too.
i am having trouble in cryopreserving the HT-29 cells. i use 5% DMSO and 95% FBS to freeze cells but i am not able revive the cells.
Dear Imran, we use 10% DMSO in normal growth medium with 10% FBS as freezing medium. Use sequential freezing temperatures (-20C /-80C >N2) with a foam box or use a cell-freezing container with isopropyl alcohol. Defrosting the cells requires quick handling and removal of DMSO. Good luck.
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