For neuronal conversion, modified fibroblasts were plated in human esophageal fibroblast medium at a density of 3.0 × 104 cells cm−2 on microscope glass coverslips coated with Matrigel (BD) in 35 mm dish. Four days after infection, the media was changed to defined neuronal induction media including Neuronal Media (ScienCell) plus 0.5 mM Dibutyryl cyclic-AMP sodium salt (dbcAMP, Sigma), 20 ng ml−1 basic Fibroblast Growth Factor (ScienCell) with medium changes every 4 days. We also added 10 ng ml−1 human brain-derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT3; both from PROSPEC) after 2 weeks to the medium to enhance induced cell survival. Quantitative of mean % iN purity is counted by morphology, TUJ1- (early stage, before one week induction) and MAP2-positive (late stage, after one week induction) staining, and the numbers represent the percentage of iN cells at the time point of quantification. Mean % neuronal yield is determined as the percentage of MAP2+ cells in relation to the initial number of plated fibroblasts at 3 week after induction.
Dibutyryl cyclic amp sodium salt dbcamp
Manufactured by Merck Group
Dibutyryl cyclic-AMP sodium salt (dbcAMP) is a chemical compound used in research applications. It is a cell-permeable analog of the second messenger cyclic adenosine monophosphate (cAMP). dbcAMP can be used to study the effects of cAMP signaling in various cellular systems.
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Lab products found in correlation
2 protocols using dibutyryl cyclic amp sodium salt dbcamp
1
Neuronal Conversion of Fibroblasts
2
Cryopreserved Cell Differentiation Protocol
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After 581 and 1006 days (CMK6 SFF), or 1183 days (CMK970) of storage, the cryopreserved cells were thawed and plated onto 24-well poly-L-ornithine/laminin-coated culture plates in DMEM/F12 supplemented with 20 ng/mL FGF2, 200 ng/mL SHH, 20 ng/mL PDGF-AA, 1 × B-27 Supplement without vitamin A, 1 × N-2 Supplement, 100 units/mL penicillin, and 100 μg/mL streptomycin at 2.5 × 105 cells per well. After 1 or 2 days, the medium was removed and replaced with DMEM/F12 with 60 ng/mL 3,3′,5-triiodo-L-thyronine (T3; Sigma-Aldrich), 100 μM dibutyryl cyclic-AMP sodium salt (dbcAMP; Sigma-Aldrich), 200 ng/mL SHH, 100 ng/mL Noggin (R&D Systems), 100 ng/mL insulin-like growth factor-I (IGF-1; R&D Systems), 10 ng/mL neurotrophin-3 (NT-3; R&D Systems), 1 × B-27 Supplement without vitamin A, 1 × N-2 Supplement, 100 units/mL penicillin, and 100 μg/mL streptomycin. Half of the media were changed twice weekly.
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