Seap reporter gene assay chemiluminescent kit

Secreted embryonic alkaline phosphatase (SEAP) secretion assays were performed as previously described (Myeni et al., 2013 (link)) with the following modifications: HeLa cells were seeded in 24-well plates to 70% confluency and co-transfected using FuGENE® 6 with plasmids expressing various truncations of BspB (300 ng DNA) and the secreted embryonic alkaline phosphatase (200 ng DNA; pSEAP2-control vector, Clontech); 500 μl of supernatant were collected and cells were lysed in 500 μl of DMEM containing 0.2% Triton X-100. Samples were centrifuged at 100 × g for 5 min to remove cell debris and SEAP activity was measured in triplicate wells, using the SEAP Reporter Gene Assay Chemiluminescent kit (Roche Cat# 11779842001). Data are represented as SEAP secretion index, which is the ratio of extracellular SEAP activity to intracellular SEAP activity, normalized to that of cells transfected with empty pCMV-HA control vector. Data are from 3 independent repeats, each performed in triplicate.

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.