I am intending to use TGF-beta receptor 1 antibody as a blocker in cultured fibroblasts treated with TGF-b. In general, to assess the FMT (fibrobllast-to-myofibroblast transition), cells are incubated with TGF-b for 5-7 days (M. Michalik et al. Respiratory Medicine, 2011). My first question is whether I should pre-incubate the cells with the antibody for few hours then give TGF-b for 5-7 days, or I should keep the antibody the whole time while TGF-b is present in the media? If yes, won't that cause the antibody to become dysfunctional?
My second question is, in case I am treating the cells with a drug, what is the protocol? Antibody first, then drug, then TGF-b? I am willing to see whether the TGF-b receptor blockade can affect my drug's activity.
Hi Bashir,
First, I strongly recommend you to incubate your cells with different amount of TGFB1 (0, 0,5, 1, 5 and 10 ng/ml) at a unique time point (e.i. 48 hours). Then, you should incubate them at different time points (0, 12, 24, 48 & 62 hours) at 5 ng/ml or the concentration you determined above. After these set up you could pre-incubate the cells with the antibody (1 hour and 30 minutes prior to the treatment with TGFB1). In such case, TGFB treatment must be done in the same media of the antibody without removing the media (usually at DMEMF10-10% SFB or 5%). In my opinion 5-7 days with TGFB1 (or with the antibody-will be dysfuntional after 5 days) is too much to see a any response mainly due to changes in proliferative rate of the fibroblast mora than a TGFB response itself (myofibroblast).
Second, your secuential step would be: antibody(- 1 hour)...Drug (-30 min.) and finally TGFB (0 min).
Good luck,
Hi,
I agree 5-7 days seems too long. We have always treated fibroblasts with 2µg/ml of TGFβ for 72 hours to differentiate them.
What fibroblasts are you using? If you have a marker such as SMA (Smooth muscle actin) that would be up regulated following treatment with TGFβ you can be sure that the antibody is blocking the effect. I would suggest to start with an hour pre-treatment and then add the TGFβ in without changing the media.
It's not clear what you are wanting to do with the drug. As if you just wanting to see the effect the blockade of the receptor has on your drug I shouldn't have thought you would need to then add the TGFβ.
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