Alk1 fc chimera protein

Isolation of colonic crypts and subsequent human primary IEC culture were performed as we reported previously17, 18. In brief, colonic mucosa was incubated in the isolation buffer17, 18 for 75 minutes at room temperature. The tissue was rinsed with Phosphate Buffered Saline (PBS) and vigorously shaken by hand to release the crypts. The released crypts were placed into culture immediately or suspended in expansion medium (EM)18 containing 20% FBS and 10% dimethyl sulfoxide (DMSO) to store at -80˚C until use.
For two-dimensional (2D) culture, isolated crypts were directly spread on 48-well plates coated with collagen hydrogel17. Cells were cultured in EM with 10mM Y-27632 (Selleckchem, TX, USA) during the initial 48 hours. The medium was changed every 48 hours. Every 6 days, cells were dissociated in collagenase type IV followed by Accutase (Stemcell Technologies, BC, Canada) and the fragmented cells were passaged to a new collagen-coated plate.
In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling19.

Isolation of colonic crypts and subsequent human primary IEC culture were performed as we reported previously17, 18. In brief, colonic mucosa was incubated in the isolation buffer17, 18 for 75 minutes at room temperature. The tissue was rinsed with Phosphate Buffered Saline (PBS) and vigorously shaken by hand to release the crypts. The released crypts were placed into culture immediately or suspended in expansion medium (EM)18 containing 20% FBS and 10% dimethyl sulfoxide (DMSO) to store at -80˚C until use.
For two-dimensional (2D) culture, isolated crypts were directly spread on 48-well plates coated with collagen hydrogel17. Cells were cultured in EM with 10mM Y-27632 (Selleckchem, TX, USA) during the initial 48 hours. The medium was changed every 48 hours. Every 6 days, cells were dissociated in collagenase type IV followed by Accutase (Stemcell Technologies, BC, Canada) and the fragmented cells were passaged to a new collagen-coated plate.
In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling19.