The procedure to generate neural progenitor cells (NPCs) was adapted from Reinhardt et al. [49 (link)] and is described in detail in Grochowska et al. [11 (link)]. Briefly, iPSCs were detached and cultured as embryoid bodies in suspension in a shaker. Neural induction was initiated by promoting Wnt and Shh signalling with the addition of SB431542 (Tocris, 1614), dorsomorphin (Abcam, ab120843), CHIR (Sigma-Aldrich, SML1046), and Purmorphamine (Stem Cell Technologies, 72,202). Embryoid bodies showing neuroepithelial development were selected, dissociated, and plated on Matrigel® (Corning, 356238)-coated plates as NPCs. From the sixth passage, NPCs were kept in NPC medium, consisting of N2B27 medium (DMEM/F-12—Neurobasal in 1:1 ratio, 1% B27 w/o Vitamin A, 0.5% N2, 1% Penicillin–Streptomycin, all from Thermo Fisher Scientific) supplemented with 3 μM CHIR, 200 μM ascorbic acid (AA, Sigma-Aldrich, A92902), and 0.5 μM Smoothened Agonist (SAG, Abcam, ab142160). Cells were kept on Matrigel-coated plates, refreshed every other day, and split with accutase (Sigma-Aldrich) in a 1:10–1:20 ratio.
Dorsomorphin
Manufactured by Abcam
Sourced in United Kingdom, United States
Dorsomorphin is a small molecule that functions as a selective inhibitor of the bone morphogenetic protein (BMP) signaling pathway. It acts by blocking the activities of BMP type I receptors, including ALK2, ALK3, and ALK6.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
A769662, by Abcam (3 mentions)
Sb431542, by Miltenyi Biotec (3 mentions)
Bdnf and nt3, by Fujifilm (3 mentions)
Y 27632, by Selleck Chemicals (3 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (3 mentions)
Compound c, by Abcam (2 mentions)
B 27 supplement a, by Thermo Fisher Scientific (2 mentions)
Neurobasal a, by Thermo Fisher Scientific (2 mentions)
Laminin, by Merck Group (2 mentions)
Ldn193189, by Merck Group (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Celltiter glo, by Promega (2 mentions)
Prism 6, by GraphPad (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glutamax l glutamine supplement, by Thermo Fisher Scientific (2 mentions)
Bfgf and egf, by Thermo Fisher Scientific (2 mentions)
Sb431542, by Bio-Techne (1 mentions)
Purmorphamine, by STEMCELL (1 mentions)
Dmem f12 neurobasal, by Thermo Fisher Scientific (1 mentions)
24 protocols using dorsomorphin
1
Efficient Neural Progenitor Cell Generation
2
AMPK Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology. Antibodies against phospho-ACC (Ser79) (CST3661), phospho-AMPKα (Thr172) (CST2535) and total AMPK (CST2532) were purchased from Cell Signaling Technology. Bis-Tris 4–12% polyacrylamide gels (345–0123), MES running buffer (161–0789) and horseradish peroxidase secondary antibody conjugate (170–6515) were obtained from Bio-Rad. Polyvinylidene Fluoride (PVDF) Immobilon-P membrane (IPVH00010) was manufactured by Merck Millipore. Protein molecular weight marker (RPN800E) was purchased from GE Healthcare Life Sciences, while enhanced chemiluminescence (ECL) reagent (32209) and BCA protein assay were purchased from Life Technologies. Metformin was obtained from Calbiochem (Merck Millipore), 2-deoxy-D-glucose from Sigma-Aldrich and Compound C (Dorsomorphin) and A-769662 from Abcam. All other reagents, unless otherwise specified, were purchased from Sigma-Aldrich or Merck Millipore.
3
Osteogenic Differentiation Assay with Dorsomorphin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The BMP/Smad signaling inhibitor dorsomorphin (Abcam) was first dissolved in dimethyl sulfoxide (DMSO, Sigma) and then diluted in the medium described below (1:1 000). DMSO diluted in medium (1:1 000) was used as the vehicle. Similar to the procedure used for the osteogenic differentiation assay, BMSCs were cultured in rhBMP2-supplemented osteogenic medium (αMEM supplemented with 10 mmol·L−1 β-GP, 50 μg·mL−1 AA2P, 10 nmol·L−1 DXA, 100 ng·mL−1 rhBMP2, 10% FBS, and 1% A/A) containing DMSO (vehicle) or dorsomorphin (4 μmol·L−1). Real-time PCR and ALP staining were performed on day 7.
4
Characterization of cinnamic acid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Trans-cinnamic acid (99% purity, Figure 1 A) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). BRL 37344 and L-748.337 were purchased from Tocris Bioscience (Bristol, UK). AICAR was purchased from TCI (Chuo-ku, Tokyo, Japan). Dorsomorphin was purchased from Abcam (Cambridge, UK). All other chemicals used in this study were of analytical grade.
5
Modulating TGFβ and BMP Signaling
6
Cortical Spheroid Differentiation from hESCs/hiPSCs
7
Isolation and Synthesis of Mandelalides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The recollection and isolation of natural product mandelalides from Algoa Bay, South Africa and total syntheses of mandelalides A and L have been described previously [4 (link),9 (link),60 (link)]. NMR data for mandelalides tested here are included in [4 (link)] (natural products) and 61 (synthetic products), and were acquired at the start of this study; integrity of all mandelalide compounds was also routinely confirmed by LC-MS. Oligomycin A was purchased from Santa Cruz Biotech (Dallas, TX, USA). Dorsomorphin was purchased from Abcam (Cambridge, UK). Paclitaxel and salinosporamide A (marizomib) were purchased from Sigma Aldrich (St. Louis, MO, USA). Erlotinib was purchased from Enzo Life Sciences (Farmingdale, NY, USA). All chemicals were reconstituted in 100% DMSO, aliquoted and stored in amber borosilicate glass vials at −20 °C for use in biological studies. Cell culture-grade DMSO was used as the vehicle for all treatments, and final concentrations of DMSO for in vitro experiments never exceeded 0.1%. Primary and secondary antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA). Specific codes were as follows: AMPK (#5831), pAMPKα T172 (#50081), ACC (#3676), pACC S79 (#11818) and GAPDH (#5174). General laboratory reagents were from VWR International (Radnor, PA, USA).
8
Osteogenesis of Diabetic BMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMSCs were induced for osteogenesis following our previously published protocol 28 . Briefly, cells were trypsinized, seeded at a density of 5,000 cells per cm2, and induced for osteogenesis by medium composed of low‐glucose DMEM, 10% FBS, 1% penicillin and streptomycin, 10 mM β‐glycerophosphate, 50 μg/ml μM l ‐ascorbic acid‐2‐phosphate, 0.1 μM dexamethasone, and 10 nM 1α,25‐dihydroxyvitamin D3 (Sigma–Aldrich, St. Louis, MO). The induction medium was changed every 3 days.
To determine if TGFB and/or bone morphogenetic protein (BMP) signaling involved in the regulation of osteogenesis of BMSCs are affected by diabetes, the cell from T1DM (T1DM‐BMSCs) and non‐T1DM donors (non‐T1DM‐BMSCs) were treated with 5 μM of dorsomorphin, an inhibitor of BMP type 1 receptor kinase, and/or 5 μM of SB431542, an inhibitor of TGFB receptor kinase (Abcam, Cambridge, MA) during 21‐day osteogenic differentiation.
To determine if TGFB and/or bone morphogenetic protein (BMP) signaling involved in the regulation of osteogenesis of BMSCs are affected by diabetes, the cell from T1DM (T1DM‐BMSCs) and non‐T1DM donors (non‐T1DM‐BMSCs) were treated with 5 μM of dorsomorphin, an inhibitor of BMP type 1 receptor kinase, and/or 5 μM of SB431542, an inhibitor of TGFB receptor kinase (Abcam, Cambridge, MA) during 21‐day osteogenic differentiation.
9
BMP Pathway Inhibition Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Overall, 100 mM stock solution of IM (Stratech) was prepared in water and stored at 4 °C; BMP pathway inhibitors LDN-193189 (LDN) (Cellagen technology) and Dorsomorphin (DOR) (Abcam) were prepared using DMSO and 10 mM stocks stored at −20 °C. Inhibitors were diluted in complete media as required. LDN interrupts the BMP pathway through inhibiting AVCR1 (ALK2) and BMPR1A (ALK3), receptor phosphorylation, whereas DOR inhibits AVCR1 (ALK2), BMPR1A (ALK3), BMPR1B (ALK6), thereby preventing phosphorylation of SMADs 1, 5 and 8, and transcription of downstream genes.
10
Metabolic Regulation in HeLa Cells
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!