Recombinant transforming growth factor β1 tgf β1

Collagenase perfusion used to isolate cultures of primary hepatocytes from mouse livers as described in detail previously^{[1 (link)]}. Primary hepatocytes were cultured in Williams’ medium E supplemented with 10% fetal bovine serum and Penicillin-Streptomycin. Following a 3-hour period of cell attachment, medium with unattached cells were removed, and fresh serum-free Williams’ E medium supplemented with Penicillin-Streptomycin was added. Following a 16 hour incubation, hepatocyte cultures were maintained in an environment containing either room air or 1% oxygen for the indicated times in a NAPCO CO2 7000 cell culture incubator (NAPCO Precision, Winchester, VA). These environments were balanced with nitrogen and contained 5% CO2.
For studies with SB-431542, hepatocytes were treated with either vehicle (dimethyl sulfoxide) or 10 μM SB-431542 (Tocris, Ellisville, MO) for 30 minutes prior to culturing the cells in either room air or 1% oxygen or treating the cells with 5 ng/ml recombinant transforming growth factor-β1 (TGF-β1) (R&D Systems, Minneapolis, MN). For studies with latent TGF-β1, hepatocytes were treated with 20 ng/ml latent TGF-β1 (R&D Systems), 30 minutes prior to hypoxia exposure.