Rat SCs from sciatic nerves of newborn rats (1–2 days-old) were isolated as described previously52 (link). SCs were grown routinely in DMEM/10% FBS (Life Technologies), supplemented with 10 ng/ml Neuregulin 1 (Nrg1; R&D Systems), and 5 μM forskolin (Sigma), plus L-glutamine and penicillin/streptomycin, hereafter termed SC proliferation medium. Cells between passages 2 and 6 were used in all experiments. >95% SC purity was achieved, assessed by Sox10 and S100β staining. To initiate differentiation, SCs were washed 4 times with DMEM and then cultured in differentiation medium containing DMEM/0.5% FBS and 1 mM dibutyl cyclic AMP (Sigma) with L-glutamine and penicillin/streptomycin, for the length of time indicated in the text, depending on the assays used. All tissue culture containers and coverslips were coated with 50 μg/ml poly-L-lysine (Sigma) in PBS for at least 30 min at room temperature and then rinsed in distilled water. Purified rat SCs seeded on coverslips were fixed in 4% paraformaldehyde (PFA) for 20 minutes and washed in 1x PBS 4 times prior to immunofluorescence staining.
Dibutyl cyclic amp
Manufactured by Merck Group
Dibutyl cyclic AMP is a laboratory reagent used in biochemical research. It is a cyclic adenosine monophosphate (cAMP) analogue with two butyl groups attached. cAMP is an important second messenger in cellular signaling pathways, and Dibutyl cyclic AMP can be used to mimic or modulate cAMP-dependent processes in experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Forskolin, by Merck Group (4 mentions)
Poly l lysine (pll), by Merck Group (3 mentions)
Dmem 10 fbs, by Thermo Fisher Scientific (2 mentions)
Neuregulin 1 nrg1, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Retinoic acid, by Fujifilm (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Fujifilm (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Polylysine, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Neuregulin 1, by R&D Systems (1 mentions)
Dmem high glucose, by R&D Systems (1 mentions)
Dmem high glucose, by Merck Group (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Ara c, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by R&D Systems (1 mentions)
6 protocols using dibutyl cyclic amp
1
Isolation and Differentiation of Rat Schwann Cells
2
Motoneuron Induction from Neural Stem Cells
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Suspended NPCs (NS1) were used for MN induction as described previously [28 ], with minor modifications. In short, mature neurospheres (>300 μM) were treated with TrypLE Select to make single-cell suspensions, count the cell number, and start adherent culturing in KBM with micronutrient-specific supplements. The supplements included 2% vitamin B27, 1% N2 (Gibco, cat. 17,502–048), 10 ng/ml recombinant human brain-derived neurotrophic factor (BDNF) (R&D system, cat. 248-BD), 10 ng/ml recombinant human glial cell line-derived neurotrophic factor (GDNF) (R&D system, cat. 212-GD), 50 ng/ml small molecule recombinant human Sonic Hedgehog (hSHH) (R&D system, cat. 1845-SH), 200 ng/ml ascorbic acid (Wako chemicals, cat. 012–04802), 1 mM dibutyl cyclic AMP (Sigma–Aldrich, cat. D0627), 10 ng/ml recombinant human insulin-like growth factor-1 (R&D system, cat. 291-G1), and 50 nM retinoic acid (Wako chemicals, cat, 182–01116). The cells were seeded on poly-L-ornithine (Sigma–Aldrich, cat. P3655) and laminin (Invitrogen, cat. 23,017–015)-coated cell desk LF1 (Sumilon, cat, MS92132) or culture plates. The culture medium was changed with the same medium on alternate days. When the color of the medium was altered in one day, the medium was changed every day until 4 weeks (28 days) of culture.
3
Isolation and Culture of Rat Sciatic Nerve Schwann Cells
4
Isolation and Differentiation of Rat Schwann Cells
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Rat SCs from sciatic nerves of newborn rats (1–2 days-old) were isolated as described previously64 (link). SCs were grown routinely in DMEM/10% fetal bovine serum (FBS) (Life Technologies), supplemented with 10 ng ml−1 Neuregulin 1 (R&D Systems), and 5 μM forskolin (Sigma), plus L-glutamine and penicillin/streptomycin, hereafter denoted as SC growth/proliferation medium. Cells between passages 2 and 6 were used in all experiments. More than 95% SC purity was achieved, assessed by positive SOX10 and S100β immunoreactivity. To initiate differentiation, SCs were cultured in differentiation medium containing DMEM/0.5% FBS and 1 mM dibutyl cyclic AMP (Sigma) with L -glutamine and penicillin/streptomycin, for the length of time indicated in the text, depending on the assays used. All tissue culture containers and glass coverslips (Carolina Biological cat# 633029) were coated with 50 μg ml−1 poly-L -lysine (Sigma) in phosphate buffered saline (PBS) for at least 30 min at room temperature and then rinsed with distilled water. For BrdU incorporation analysis, BrdU at 20 μM was added to purified rat SCs seeded on coverslips 24 h or otherwise indicated, prior to fixation in 4% (w/v) paraformaldehyde (PFA).
5
Isolation and Differentiation of Rat Schwann Cells
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Rat SCs from sciatic nerves of newborn rats (1–2 days-old) were isolated as described previously52 (link). SCs were grown routinely in DMEM/10% FBS (Life Technologies), supplemented with 10 ng/ml Neuregulin 1 (Nrg1; R&D Systems), and 5 μM forskolin (Sigma), plus L-glutamine and penicillin/streptomycin, hereafter termed SC proliferation medium. Cells between passages 2 and 6 were used in all experiments. >95% SC purity was achieved, assessed by Sox10 and S100β staining. To initiate differentiation, SCs were washed 4 times with DMEM and then cultured in differentiation medium containing DMEM/0.5% FBS and 1 mM dibutyl cyclic AMP (Sigma) with L-glutamine and penicillin/streptomycin, for the length of time indicated in the text, depending on the assays used. All tissue culture containers and coverslips were coated with 50 μg/ml poly-L-lysine (Sigma) in PBS for at least 30 min at room temperature and then rinsed in distilled water. Purified rat SCs seeded on coverslips were fixed in 4% paraformaldehyde (PFA) for 20 minutes and washed in 1x PBS 4 times prior to immunofluorescence staining.
6
Schwann Cell Isolation and Differentiation
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Sciatic nerves were isolated from P0-P1 rats, and digested with 1 mg/ml collagenase A (Sigma). Schwann cells were plated into poly-D-lysine (Sigma) coated dish and grown in DMEM High Glucose (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1 mM Ara-C (Sigma) for 3 days. After passage, Schwann cells were cultured with proliferation medium (DMEM High Glucose supplemented with 10% fetal bovine serum, 10 ng/ml NRG1 (R&D Systems), 5 μM forskolin (Sigma), 1% L-glutamine (Hyclone, Cat# SH30034) and 1% penicillin/streptomycin). To differentiate, Schwann cells were cultured in differentiation medium (DMEM High Glucose supplemented with 0.5% fetal bovine serum and 1 mM dibutyl cyclic AMP (Sigma) with 1% L-glutamine and 1% penicillin/streptomycin).
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