Quantifying Serum TGF-β Bioactivity in Mice

Blood was drawn from mice by submandibular bleeding before treatment (week 18), during treatment (week 20) and at the experimental end-point (week 22) and centrifuged at 1000 xg for 10 min to obtain serum, which was frozen at −80 °C until further use. A TGF-β bioassay was used to measure serum levels of bioactive TGF-β. Therefore, MFB-F11 reporter cells were used according to the protocols published by Tesseur et al. [23 (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 serum samples were added in 10 μl volume. For SEAP assay, a SEAP Reporter Gene Assay Chemiluminescent kit (Roche) was used according to the manufacturer’s recommendations.