Human astrocytes were generated from neuroepithelial-like stem (NES) cells, produced from human induced pluripotent stem cells (iPSCs, Cntrl9 cell line) [23 (link), 24 (link)]. The cells were cultured in Advanced DMEM/F12 (ThermoFisher, 12634–010) supplemented with 1% penicillin streptomycin (ThermoFisher, 15140–122), 1% B27 supplement (ThermoFisher, 17504–044), 1% non-essential amino acids (Merc Millipore) and 1% L-glutamine (ThermoFisher, 25030–024). The following factors were added to the medium just before use: basic fibroblast growth factor (bFGF) 10 ng/ml (ThermoFisher, 13256029), heregulin β-1 10 ng/ml (Sigma-Aldrich, SRP3055), activin A 10 ng/ml (Peprotech, 120-14E) and insulin-like growth factor 1 (IGF-1) 200 ng/ml (Sigma-Aldrich, SRP3069). Additionally, 20 ng/ml ciliary neurotrophic factor (CNTF; ThermoFisher, PHC7015) was added to the medium for the last 2 weeks of culture. Cells were passaged using trypsin 0.05% EDTA 0.2 g/l (Life Technologies) and were used for experiments directly after differentiation.
Heregulin β1
Manufactured by Merck Group
Sourced in United States
Heregulin β1 is a recombinant protein that functions as a ligand for the human epidermal growth factor receptor 3 (HER3) and human epidermal growth factor receptor 4 (HER4). It is commonly used in cell culture and biochemical applications to study cell signaling pathways involving the HER3 and HER4 receptors.
Automatically generated - may contain errors
Lab products found in correlation
Forskolin, by Merck Group (4 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Activin a, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Trametinib, by Adooq (1 mentions)
Rapamycin, by Santa Cruz Biotechnology (1 mentions)
Bortezomib, by Adooq (1 mentions)
Sch772984, by Adooq (1 mentions)
Mk 2206, by Adooq (1 mentions)
Bovine pancreatic trypsin, by Merck Group (1 mentions)
Perchloric acid, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Dialysis membrane, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
6 protocols using heregulin β1
1
Generation of Human Astrocytes from iPSCs
2
Modulation of Intracellular Signaling Pathways
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Heregulin β-1 (Sigma, H7660) was used at a working concentration of 20 ng.mL−1. For lysosome and proteasome inhibition, cells were treated for 4 h with 50 nM of bafilomycin A1 (Calbiochem, 196000) and 1 µM of bortezomib (Adooq Bioscience, A10160), respectively. CHX (Sigma, 01810) was used for translation inhibition at 25 µg.mL−1. For targeted inhibition of intracellular signaling proteins, trametinib (Adooq Bioscience, JTP-74057, [100] nM), rapamycin (Santa Cruz Biotechnology, sc-3504A, [100] nM), buparlisib (Adooq Bioscience, AT11016, [500 nM]), selumetinib (Adooq Bioscience, A10257, [1 µM]), SCH772984 (Adooq Bioscience, A12824, [1] µM) and MK-2206 (Adooq Bioscience, A10003, [2] µM) were used for the indicated time points.
3
Biomaterials for Cell Culture Applications
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Polyvinyl alcohol (molecular weight (MW) 124,000–184,000 Da), 70% hydrochloric acid, acetonitrile, elementary bromine, perchloric acid, sodium bicarbonate, bovine pancreatic trypsin, poly-L-lysine, Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum, penicillin/streptomycin solution, gentamicin solution, Heregulin β1, Forskolin, ethylenediaminetetraacetic acid, [3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide] and dialysis membranes (cut-off 8000 Da) were purchased from Sigma-Aldrich (S. Louis, Missouri, USA). Potassium permanganate, ascorbic acid, acetic acid, 2-propanol, 100% ethanol, sodium iodide, sodium cyanoborohydride and dimethyl sulfoxide were purchased from Carlo Erba (Milan, Italy).
4
Culturing Rat Sciatic Nerve Microtissues
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Five 3-day-old SD rats were intraperitoneally injected with 2% sodium pentobarbital solution before operation. The sciatic nerve was removed and cut into 1-mm3 pieces using microscissors (Jinzhong, Shanghai, China). Nerve microtissue was cultured in Dulbecco’s modified Eagle medium/F12 (Corning, Christiansburg, VA, USA) supplemented with 10% fetal bovine serum (Corning), 10 ng/mL heregulin-β1 (Sigma-Aldrich, St. Louis, MO, USA) and 2 mM forskolin (Sigma-Aldrich) in six-well plates in an incubator (Heal Force, Shanghai, China) containing 5% CO2 at 37°C. Then, after 1, 3, 5 or 7 days in culture, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining were performed (Figure 1
5
Isolation and Purification of Rat Schwann Cells
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As previously described, primary SCs were isolated from the sciatic nerve of neonatal Sprague-Dawley (SD) rats [40 (link)]. Briefly, the sciatic nerve was dissected and treated with collagenase and trypsin. Collected cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin and streptomycin (Invitrogen), 2 μM forskolin (Sigma, St. Louis, MO, USA), and 10 ng/ml heregulin β1 (Sigma) till confluence. Cells were then treated with anti-Thy1.1 antibody (Sigma) and rabbit complement (Invitrogen) to remove fibroblasts. Purified SCs were grown in a culture medium containing DMEM, 10% FBS, and 1% penicillin and streptomycin (Invitrogen) in a humidified 5% CO2 incubator at 37 °C. Primary SCs were passaged for no more than two passages before use. The purity of SCs was identified by immunocytochemical staining using the primary antibody rabbit anti-S100 (1:200 dilution, Abcam), and the second antibody goat anti-rabbit IgG-Cy3 (1:100 dilution, Abcam), and nucleus were labeled with 4’,6-diamidino-2-phenylindole (DAPI) (SouthernBiotech).
6
Organoid Culture from Surgical Samples
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Freshly obtained surgical samples were digested first with a mixture of collagenase and dispase, followed by TrypLE (ThermoFisher). After passing through a mesh, cells were embedded in Matrigel (Corning) and cultured in four different organoid media formulations in parallel. These formulations have the same base media containing the growth factors EGF, Noggin (Sigma), R-spondin (Sigma), and FGF10 (ThermoFisher) as well as N-acetylcysteine (ThermoFisher), nicotinamide, Y-27632 (Rho kinase inhibitor), A83-01 (TGF-β signaling inhibitor), N2, and B27 (all from Sigma). Additional components of these media include forskolin (adenylyl cyclase activator), CHIR99021 (Wnt activator), gastrin I, prostaglandin E2, FGF2, hydrocortisone, and heregulin β1 (all from Sigma). The cells that demonstrated the most robust growth were further expanded, and fractions of cells were harvested for histology, DNA and RNA isolation, or cryopreserved at low passage numbers.
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