DMEM–Dulbecco's Modified Eagle Medium

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	CHO-K1 cells were cultured in DMEM/F-12 (Invitrogen), and Bosc23 and B16-F1 cells were cultured in DMEM (Invitrogen), all supplemented with 10% fetal calf serum and 100 μg/ml penicillin/streptomycin. Cells were transfected with plasmids encoding secreted ShhN, ShhNp, or sAP-ShhN using PolyFect (Qiagen). Cells were grown for 36 h, washed with phosphate-buffered saline, and incubated in DMEM or serum-free DMEM for various time periods in the presence or absence of stimulators or inhibitors of shedding, followed by ultracentrifugation at 210,000 × g for 60 min to remove cell debris, including membrane-tethered ShhNp.

Protocol tips

CHO-K1 cells were cultured in DMEM/F-12 (Invitrogen), and Bosc23 and B16-F1 cells were cultured in DMEM (Invitrogen), all supplemented with 10% fetal calf serum and 100 μg/ml penicillin/streptomycin. Cells were transfected with plasmids encoding secreted ShhN, ShhNp, or sAP-ShhN using PolyFect (Qiagen). Cells were grown for 36 h, washed with phosphate-buffered saline, and incubated in DMEM or serum-free DMEM for various time periods in the presence or absence of stimulators or inhibitors of shedding, followed by ultracentrifugation at 210,000 × g for 60 min to remove cell debris, including membrane-tethered ShhNp.

Publication protocol

CHO-K1 cells were cultured in DMEM/F-12 (Invitrogen), and Bosc23 and B16-F1 cells were cultured in DMEM (Invitrogen), all supplemented with 10% fetal calf serum and 100 μg/ml penicillin/streptomycin. Cells were transfected with plasmids encoding secreted ShhN, ShhNp, or sAP-ShhN using PolyFect (Qiagen). Cells were grown for 36 h, washed with phosphate-buffered saline, and incubated in DMEM or serum-free DMEM for various time periods in the presence or absence of stimulators or inhibitors of shedding, followed by ultracentrifugation at 210,000 × g for 60 min to remove cell debris, including membrane-tethered ShhNp.

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