For growth factor removal, mixed strain (129 × BL6) of wild-type newborn mouse skin was used as described to derive primary keratinocytes, which were expanded and used for experiments (between passages 4–6)47 (link). Cells were first grown to confluency. Then, cells were washed once with PBS and added serum-free media containing either recombinant BMP4 (R&D Systems) between 1 and 2 nM or its carrier (4 mM HCl in 0.1%BSA) only. Cells incubated for 6 and 12 h were harvested for RNA extractions.
Recombinant bmp4
Manufactured by R&D Systems
Sourced in United States
Recombinant BMP4 is a purified recombinant protein produced in HEK293 cells. BMP4 is a member of the transforming growth factor beta (TGF-β) superfamily and plays a role in bone and cartilage development.
Automatically generated - may contain errors
Lab products found in correlation
Brain derived neurotrophic factor (bdnf), by R&D Systems (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Gfap mouse monoclonal, by Merck Group (1 mentions)
U0126, by Bio-Techne (1 mentions)
Recombinant tgf β, by R&D Systems (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
H1 hesc, by WiCell (1 mentions)
Tgfb3, by R&D Systems (1 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
Chir99021, by Selleck Chemicals (1 mentions)
8 protocols using recombinant bmp4
1
BMP4-Induced Keratinocyte Response
2
Serum-free embryoid body differentiation
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Serum-free embryoid body quick (sfEBq) aggregation and initial differentiation were performed as previously described (Eiraku et al., 2011 (link); Koehler and Hashino, 2014 (link)). In brief, 3,000 mESCs per well were aggregated on day 0 at the bottom of U-shaped low adhesion 96-well plates in ectoderm differentiation medium [G-MEM with 1.5% knockout serum replacement (KSR), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 1 mM penicillin/streptomycin, and 1 mM 2-mercaptoethanol]. The following day (day 1), half of the medium was exchanged for differentiation medium containing 2% Matrigel (v/v final concentration). On day 3, recombinant BMP-4 (R&D) 10 ng/ml together with 1 μM of transforming growth factor beta (TGF-β) inhibitor SB 43-1542 (Stemgent) was supplemented to the medium. At day 4½to day 5, 25 ng/ml FGF-2 (Peprotech), and 1 μM BMP inhibitor LDN-193989 (Stemgent) were added to the culture.
Organoids were then plated on Matrigel-coated well plates or glass slides. Matrigel was diluted for coating 1:4 or 1:10 depending on the experiment. On day 8, after assessing attachment of the organoids to the culture plate, medium was changed to OSN medium, containing: DMEM/F12, B27, N2, NT3 (5 ng/ml) (Peprotech Cat. No. 450-03), and BDNF (5 ng/ml) (R&D Cat 248-BD). Medium was changed every other day until termination of the experiment.
Organoids were then plated on Matrigel-coated well plates or glass slides. Matrigel was diluted for coating 1:4 or 1:10 depending on the experiment. On day 8, after assessing attachment of the organoids to the culture plate, medium was changed to OSN medium, containing: DMEM/F12, B27, N2, NT3 (5 ng/ml) (Peprotech Cat. No. 450-03), and BDNF (5 ng/ml) (R&D Cat 248-BD). Medium was changed every other day until termination of the experiment.
3
Neural Stem Cell Differentiation Protocol
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TUJ1 rabbit monoclonal was from Covance (Princeton, NJ, USA product number MRB-435P). GFAP mouse monoclonal was from Sigma-Aldrich (Oakville ON Canada, product number G3893). Nestin mouse monoclonal antibody (product number MAB1259), recombinant BMP4 and recombinant TGFβ were from R&D Systems (Minneapolis MN, USA). p21 mouse monoclonal antibody, pSMAD2 rabbit monoclonal antibody and SMAD2 rabbit monoclonal antibody were from Cell Signaling Technology (Danvers MA, USA, product numbers DCS60, 138D4 and D43B4). PML mouse monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz CA, USA, product number PG-M3). LSKL peptide was from AnaSpec (Fremont CA, USA). U0126 was from Tocris Bioscience (Bristol UK).
4
Llama Immunization with Recombinant BMP4
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Example 1
Recombinant BMP4 was purchased from R&D systems and treated according to the manufacturer protocol. BMP4 was dissolved in 4 mM HCl at 0.1 mg/ml and 50 μg was diluted in PBS (1 ml), mixed with 1 ml adjuvant Stimune and injected intramuscular in two llamas (llama#18-2 and llama#31-2) according to the following schedule: Immunizations on day 0, day 14, day 28 and day 35. Blood was taken at day 0 (preimmune) and day 43 (immune). Large blood uptake at day 43 was used for PBL isolation and RNA extraction.
Immune Responses
Immune responses directed against BMP4 were measured in the serum taken at day43 (end of immunizations and PBL isolation) and compared to signal measured in serum of day 0 (preimmune). BMP4 was coated at 200 ng/well of a Maxisorp plate and ELISA was performed with serial dilutions of preimmune serum (day0) and immune serum (day43). Immune sera of both llama showed binding to immobilized BMP4 in contrast to preimmune sera (
5
BMP4 Modulates SKOV3, CAOV-3 and COV318 Cells
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SKOV3, CAOV-3 and COV318 cells were plated (2×105 cells/6 well dish) in serum-free media treated with 200ng/ml recombinant BMP4 (R&Dsystems, Pittsburg, PA) for 16 and 48hours. Cells were then processed for RT-PCR or immunoblotting.
6
Human Embryonic Stem Cell Differentiation Protocols
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H1 hESCs (Wicell) were cultured on Biomatrix in feeder-free culture conditions with mouse embryonic fibroblast-conditioned media (MEF-CM) (Tan et al., submitted for publication). Briefly, human embryonic stem medium [80% Dulbecco's modified Eagle's medium-Ham's F-12 medium, 20% Knockout serum replacement (Invitrogen), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids and 4 ng/ml human basic fibroblast growth factor (bFGF)] was incubated with mitomycin C-inactivated MEFs overnight, with an additional 4 ng/ml of bFGF before hESC feeding. Cultures were passaged every 5–7 days, before they became confluent. Differentiation was induced via the addition of specific factors into MEF-CM, along with the removal of bFGF. For trophoblast induction, cultures were incubated in MEF-CM with 100 ng/ml of recombinant BMP4 (R&D Systems) and 20 μM of FGFR-inhibitor SU5402 (Calbiochem) for 1–5 days (30 (link)). For retinoid differentiation, cultures were incubated in MEF-CM with 10 μM of synthetic retinoid EC23 (Reinnervate) for 1–5 days (31 (link)).
7
Establishing Pancreatic Cancer Cell Lines
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Human pancreatic adenocarcinoma Panc-1 and BxPC-3 cells were cultured as described previously. 16, 17 Human pancreatic adenocarcinoma SUIT-2 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin. Cells were grown in a 5% CO 2 atmosphere at 37 C. All experiments were performed on heterogenous populations of shRNA-transfected or adenovirus-infected cells. Among three TGF-b isoforms, TGF-b3 (R&D Systems, Minneapolis, MN) was used in this study. Recombinant BMP-4 was purchased from R&D Systems.
shRNA Lentivirus vectors carrying shRNA were generated as described previously. 18 pENTR4-H1 was used to insert shRNA specific for human SMAD4 and ALDH1A1 into the lentivirus vectors CSII-RfA-CG and CS-RfA, respectively. Vectors were also generated using control shRNA. The sequences of shRNAs are listed in Table 1. Lentiviruses were concentrated using Lenti-X Concentrator (Clontech, Mountain View, CA).
shRNA Lentivirus vectors carrying shRNA were generated as described previously. 18 pENTR4-H1 was used to insert shRNA specific for human SMAD4 and ALDH1A1 into the lentivirus vectors CSII-RfA-CG and CS-RfA, respectively. Vectors were also generated using control shRNA. The sequences of shRNAs are listed in Table 1. Lentiviruses were concentrated using Lenti-X Concentrator (Clontech, Mountain View, CA).
8
Trilineage Differentiation Protocols for Stem Cells
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Recombinant BMP4 (R and D Systems) was added to mTeSR at 50 ng/mL for 24 hours, starting 24 hours after aggregate seeding to induce a trilineage differentiation, 14 followed by 24 hours of imaging in mTeSR alone. CHIR differentiation was performed by adding 12 𝜇𝑀 CHIR-99021 (Selleck Chemicals) to mTeSR for 24 hours, starting 24 hours after seeding, followed by 24 hours of imaging in mTeSR only. Dual SMAD inhibition was performed by adding both 10 𝜇𝑀 SB-431542 (GlaxoSmithKline) and 0.2 𝜇𝑀 LDN-193189 (Stemgent) to mTeSR 41 during force aggregation, and maintained for 72 hours through colony adhesion and imaging. CHIR pre-treatment during dual SMAD inhibition was achieved by adding 2 𝜇𝑀 CHIR-99021 to mTeSR starting 48 hours before force aggregation, and continued through imaging (5 total days of treatment) 43 (link) .
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