Recombinant human LIF (R&D Systems), human oncostatin M (R&D Systems), mouse LIF (Miltenyi Biotec), mouse oncostatin M (R&D Systems), and human TGF-β1 (R&D Systems) were reconstituted in PBS + 0.1% bovine serum albumin (BSA) at 10–25μg/ml and aliquoted for storage at −80°C. For all experiments, mouse recombinant proteins were used on mouse cell lines, and human recombinant proteins were used on human cell lines, with the exception of human TGF-β1, which was used on all cell lines since TGF-β1 maintains approximately 99% sequence homology between human and mouse species63 (link). Prior to cytokine treatment cells were serum starved in 2% FBS overnight and cytokine treatment was made up in media containing 2% FBS.
Mouse oncostatin m
Manufactured by R&D Systems
Sourced in Japan, United States
Oncostatin M is a recombinant mouse protein that belongs to the interleukin-6 cytokine family. It is a multifunctional cytokine involved in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Human tgf β1, by R&D Systems (2 mentions)
Recombinant human lif, by R&D Systems (2 mentions)
Human oncostatin m, by R&D Systems (2 mentions)
Mouse lif, by Miltenyi Biotec (2 mentions)
Non adherent 24 well plates, by Corning (2 mentions)
Mouse igf 1, by R&D Systems (1 mentions)
Mouse pdgf aa, by Thermo Fisher Scientific (1 mentions)
Cfi plan fluor dl, by Nikon (1 mentions)
Mouse egf, by Merck Group (1 mentions)
Bz x viewer, by Keyence (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
Bz x700 all in one fluorescence microscope, by Keyence (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Mouse egf, by Thermo Fisher Scientific (1 mentions)
Human bfgf, by R&D Systems (1 mentions)
Mouse pdgf, by Thermo Fisher Scientific (1 mentions)
Human endothelial medium, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
6 protocols using mouse oncostatin m
1
Cytokine Reconstitution and Cell Treatment
2
Cytokine Reconstitution and Cell Treatment
3
Spheroid Formation from Murine Bone Marrow and Periodontal Ligament Cells
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Cells collected by the cell sorter were cultured in non-adherent 24 well plates (Corning, New York, NY, USA) (LepR/Tom+ BM cells: 4781‒9849 cells/well, LepR/Tom− BM cells: 18,642‒201,149 cells/well, LepR/Tom+ PDL cells: 83‒348 cells/well, LepR/Tom− PDL cells: 9125‒61,003 cells/well) with spheroid-forming media22 (link),27 (link)–29 (link) (1:2 ratio of DMEM/F-12 (1:1) (21331-020) and Human Endothelial Medium (11111-044) supplemented with 3.75% Chicken Extract, 0.1 mM β-ME, 1% Non-essential amino acids, 1% Antibiotic–Antimycotic, 1% N2, 2% B27, 20 ng/mL mouse PDGF-AA (all from Thermo Fisher Scientific), 20 ng/mL human bFGF (ReproCELL Inc., Kanagawa, Japan), 20 ng/mL mouse oncostatin M, 20 ng/mL mouse IGF-1 (all from R&D SYSTEMS), 20 ng/mL mouse EGF (Sigma-Aldrich, St. Louis, MO, USA)). After culturing for 14 days, spheroid-forming efficiency was determined. Fluorescence and phase-contrast images of spheroids were acquired using an All-in-One Fluorescence Microscope (BZ-X700) equipped with a BZ-X-Viewer, BZ-X Analyzer (all from KEYENCE, Osaka, Japan), a CFI Plan Fluor DL (4×/0.13), and a CFI Plan Fluor DL (10×/0.45) (both from Nikon, Tokyo, Japan).
4
Spheroid Formation from Sorted Cells
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Mouse sorted cells at 1 × 103 were transferred to non-adherent 24 well plates (Corning) with spheroid-forming media5 (link), 7 (link), 47 (link) [1:2 ratio of DMEM F12 (Gibco) and Human Endothelial Medium (Gibco) supplemented with 3.75% Chicken Extract (US Biological), 0.1 mM β-ME (Invitrogen), 1% Non-essential amino acids (Gibco), 1% Pen-strep (Gibco), 1% N2 (Gibco), 2% B27 (Gibco), 20 ng/mL human bFGF (R&D Systems), 20 ng/mL mouse PDGF (Peprotech), 20 ng/mL mouse oncostatin M (R&D Systems), 20 ng/mL mouse IGF-1 (Peprotech), 20 ng/mL mouse EGF (Peprotech)]. After 7 days, the spheroid efficiency was determined.
5
Hepatic and Cholangiocyte Differentiation Protocol
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For hepatic differentiation, 1×105 AML12 or LPS-AML12 cells were plated on collagen-coated dishes in DMEM/F12 medium containing 10% FBS, 1% penicillin/streptomycin, 20 ng/mL mouse oncostatin M (R&D Systems, MN, USA), 20 ng/mL mouse HGF (Peprotech, USA), and 10-7 M dexamethasone (Sigma) [24 (link)]. And the medium was changed every 2 days. After 1 week, cells were collected and used for other functional assays. For cholangiocyte differentiation, 1×105 cells were seeded on collagen-coated dishes in DMEM/F12 medium supplemented with 10% FBS and 20 ng/mL HGF (Peprotech, USA) for 7 days, with fresh medium provided every 2 days [24 (link)]. After induction, cells were used for other assays.
6
Scalable 3D Spheroid Culture Protocol
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To generate spheroids, we used Aggrewell400 24-well plates (Stemcell Technologies), which were pre-treated with Anti-Adherence Rinsing Solution according to the manufacturer’s instructions (Stemcell Technologies). After trypsinization, cells were resuspended in SHM+YAC without FBS, and the cell concentration was adjusted to 1–1.2 × 106 cells/mL. Then, 1 mL of the cell suspension was added to an Aggrewell400 plate well, which was pre-filled with 1 mL of SHM+YAC, and mixed thoroughly by pipetting. Then, plates were centrifuged at 100 × g for 3 min, and placed in a CO2 incubator. Following incubation for 3 days without medium change, spheroids were harvested in a 15-mL tube. After sitting for approximately 5 min, the supernatant was removed, and spheroids were resuspended in 5 mL SHM+YAC supplemented with 10 ng/mL mouse oncostatin M (R & D), and seeded to a 60 mm ultra-low attachable suspension culture plate (Corning). Then, spheroids were cultured on a rocking shaker at 15 rpm speed for 6–8 days. Half volume (2.5 mL) of the medium was removed and 2.5 mL fresh medium was replenished every 2 days.
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