Hi
I am interested to find out if TGF-beta is involved in different cell-cell communication between 3 cell types in the glomerular filtration barrier. I have search quite a number of published papers and it is not known if TGF-beta is primarily expressed in a particular cell type or all 3 cell types in this filtration barrier. Also TGF-beta can be secreted so it is not known if they can be a mediator in cross talking between the different cell types in this barrier.
how do i even go about to do this?
first and foremost my lab is limited to dealing with cell lines only.
1) probably i should see the expression (RNA and protein) status of tgf-beta in each of the 3 cell types.
2) culture each cell type individually and see if they secrete TGF-beta in media? (sounds stupid)
3) co-culture all 3 cells together but im lost as to how to investigate if TGF-beta from any of the cell types can be used to communicate with the other cell type.
I would suggest step-3 first because that's the key question that will tell you, whether there is an effect from one cell to other, and it will be cheaper test. For-3 you can use filtered conditioned media to see if there is any effect. If any effects are observed, then you have to know that it is TGF-b specific. For this use TGF-b neutralizing antibody with conditioned media to see if that effect is reversed (see TNFa model in my paper: https://www.researchgate.net/publication/233710524_Smac_mimetic_enables_the_anticancer_action_of_BCG-stimulated_neutrophils_through_TNF-_but_not_through_TRAIL_and_FasL)
If step-3 is fruitful then you'll have to do step-1 and 2 anyway. For step-2 ELISA is best but expensive if bought commercial kit. If you can get the antibody, I would recommend home made ELISA using HRP, ABTS.
Good Luck.
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