TGFβ Bioassay Protocol with Inhibitor Pretreatment

A TGFβ bioassay was used to measure levels of bioactive TGFβ based on MFB‐F11 reporter cells according to the protocols published by Tesseur et al. (Tesseur et al., 2006 (link)). Briefly, MFB‐F11 cells were seeded at 4 × 10⁴ cells/well in 96‐well flat‐bottom tissue culture plates (BD Falcon). After overnight incubation, cells were washed twice with PBS and incubated in 50 μl serum‐free DMEM supplemented with penicillin/streptomycin for 2 h before samples were added. For the SEAP assay, a SEAP Reporter Gene Assay Chemiluminescent kit from Roche was used according to the manufacturer's recommendations. For some experiments, MFB‐F11 cells were pre‐incubated at the indicated concentrations with Dynasore, Cytochalasin D or Nystatin (all purchased from Abcam) for 1 h. Cells were washed twice with PBS and the selected tested factors were added. For other experiments, MFB‐F11 cells were pre‐incubated with TGFβ inhibitors added before the start of experiments and used at the indicated concentrations for 2 h.