N2a cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/l glucose supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin (all from Life Technologies) in a humidified atmosphere containing 5% CO2 at 37 °C. To differentiate the cells, the cells were cultured with a differentiation medium (DMEM containing 4.5 g/l glucose supplemented with 2% (v/v) FBS and 2 mM N,N-dibutyladenosine 3′,5′-phosphoric acid (dbcAMP; Nacalai Tesque Inc., Kyoto, Japan)).
Dbcamp
Manufactured by Nacalai Tesque
Sourced in Japan
DbcAMP is a chemical compound used in laboratory research. It is a cyclic adenosine monophosphate (cAMP) analog that can be used to study signal transduction pathways and cellular processes in various experimental models.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (4 mentions)
Brain derived neurotrophic factor (bdnf), by Alomone (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Fujifilm (1 mentions)
Glomax, by Promega (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Doxycycline, by Fujifilm (1 mentions)
Cultureone supplement, by Thermo Fisher Scientific (1 mentions)
B 27 plus supplement, by Thermo Fisher Scientific (1 mentions)
Purmorphamine, by Merck Group (1 mentions)
Neurobasal plus medium, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
5 protocols using dbcamp
1
N2a Neuronal Cell Differentiation
2
N2a cell viability assay with cAMP
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N2a cells were seeded at 2.0 × 105 cells/ml in 96-well plates in DMEM containing 10% FBS. After transfection of each plasmid, the cells were differentiation for 48 h in low glucose (1.0 g/l glucose) DMEM supplemented with 2% FBS and 2 mM N,N-dibutyladenosine 3′,5′-phosphoric acid (dbcAMP; Nacalai tesque, Kyoto, Japan) with or without EBGP, kaempherol, or kaempheride in the presence or absence of 3-MA. The number of live cells was estimated by Cell Counting Kit-8, following the manufacturer’s instructions (Wako Pure Chemical Industries Ltd.). Briefly, reagent was added into wells and the plate was incubated at 37 °C for 4 h. The optical density of formazan was detected at 450 nm by GloMax® (Promega, Madison, WI, USA) for calculating cell viability. The wavelength of 600 nm was used as reference.
3
Feeder-free iPSC-derived Motor Neurons
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Motor neurons were generated from feeder-free iPSCs by transient expression of NIL factors using the tet-on inducible expression system. At the passage, dissociated iPSCs were seeded onto poly-L-ornithine (Merck) and Matrigel (Corning, Steuben County, NY, USA) coated culture plates and cultured in neuronal medium with the following composition: Neurobasal Plus medium (Thermo Fisher Scientific) containing 2% B27 Plus supplement (Thermo Fisher Scientific), 1% Culture One supplement (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 200 μM L-ascorbic acid (Sigma), 200 μM dbcAMP (Nacalai), 20 ng/mL BDNF (Alomone labs), 20 ng/mL GDNF (Alomone labs), and 20 ng/mL NT-3 (Alomone labs). 20 μM Y27632, 1 μg/mL doxycycline (Wako), 1 μM retinoic acid (Merck), 1 μM purmorphamine (Merck) and 0.5 μg/mL iMatrix-511 silk were added only for 5 days. Half of the medium was changed every 3 or 4 days after day 5.
4
Feeder-free Neuronal Differentiation of hPSCs
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Feeder-free cultured hPSCs were used for the induction of neuronal differentiation. The cells were dissociated using 0.5× TrypLE Select and seeded on culture dishes coated with poly-L-lysine, and iMatrix-511. To induce glutamatergic neurons, Ngn2-transduced cells were cultured in neuronal medium with the following composition: Neurobasal Plus medium containing 2% B27 Plus supplement, 1% Culture One supplement, 1% Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), 200 µM L-ascorbic acid (Merk, Darmstadt, Land Hessen, Germany), 100 µM dbcAMP (Nacalai, Kyoto, Japan), 20 ng/ml BDNF, 20 ng/ml GDNF, 20 ng/ml NT-3 (Alomone labs, Jerusalem, Israel) and 0.5 µg/mL iMatrix-511 silk. 20 µM Y27632 and 2 µg/ml Dox (FUJIFILM Wako Pure Chemical, Osaka, Japan) were added on day 0. On day 5, the medium was replaced with the fresh neuronal medium described above. For the CellROX assay and extracellular flux analyses assay, cells were maintained in the following medium after day 5: Neurobasal plus medium containing 2% B27 minus antioxidants, 1% Culture One supplement, 1% Glutamax, 20 ng/ml BDNF, 20 ng/ml GDNF, 200 µM L-ascorbic acid, 300 µM dbcAMP and 0.5 µg/ml iMatrix-511 silk. The medium was replaced every 3 days after day 5.
5
Immunostaining of Huntington's Disease
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Immunostaining was performed as reported previously. 6 In brief, HD150Q cells or HD150Q-NLS cells were differentiated with 5 mM N 6 ,2′-O-dibutyryladenosine-3′:5′-cyclic monophosphate sodium salt (dbcAMP, Nacalai, Kyoto, Japan) and induced to express tNhtt-polyQ with 1 µM Ponasterone A. After 2 days, immunocytochemistry was performed. The frozen mouse brain sections and para n-embedded human specimens were also subjected to immunohistochemistry as described previously. 13 Brie y, frozen mouse brain sections were washed twice with phosphate-buffered saline (PBS), xed for 30 min with 100% methanol, washed three times PBS, blocked for 1 h with TBST containing 2% non-fat dried milk, and incubated overnight at 4 °C with primary antibodies. Then, the sections were washed three times with TBST and incubated with secondary antibody for 2 h, and detected using Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Human brain sections were autoclaved at 121 °C for 10 min, incubated in 100% formic acid for 5 min, and incubated 1% hydrogen peroxidase for 15 min. The sections were then blocked for 1 h with TBST containing 7% goat serum, and then immunostained with primary antibodies followed by visualization using the Vectastain ABC kit.
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