Neuro-2A neuroblastoma (N2A) cells stably expressing human APP carrying the K670N/M671L Swedish mutation (N2A-APPswe) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 100U/mL streptomycin (Cellgro, Herdon, VA), and 400mg/ml G418 (Invitrogen, Carlsbad, CA) at 37°C in the presence of 5% CO2. The cells were cultured to 80 to 90% confluence in 6-well plates and then changed to fresh medium containing 50μM DL-Hcy (Sigma, St Louis, MO) with 40μM adenosine (Sigma) and 10μM erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride (Sigma), as previously described.10 (link),17 (link) Cell toxicity was monitored by measuring the amount of the lactate dehydrogenase enzyme released in the supernatant at the end of the incubation time by a colorimetric assay kit (Cell Biolabs, San Diego, CA). After 24 hours of treatment, media were collected for Aβ1–40 measurement, and cell lysates were harvested after treatment with radioimmunoprecipitation assay (RIPA) buffer for Western blotting analyses.
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Neuroblastoma Model for Alzheimer's Research
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streptomycin
DL-Hcy
adenosine
erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride
colorimetric assay kit
Adenine
Adenosine
After treatment
Assay
Buffer
Cell
Colorimetric
Eagle
Enzyme
Fetal bovine serum
G418
Human
Lactate dehydrogenase
Mutation
Neuroblastoma
Radioimmunoprecipitation analyses
Streptomycin
Western blotting assay
Corresponding Organization :
Other organizations : Temple University
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Variable analysis
independent variables
- DL-Hcy (50μM)
- Adenosine (40μM)
- Erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride (10μM)
- Aβ1–40 measurement in media
- Western blotting analyses of cell lysates
- Neuro-2A neuroblastoma (N2A) cells stably expressing human APP carrying the K670N/M671L Swedish mutation (N2A-APPswe)
- Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 100U/mL streptomycin, and 400mg/ml G418
- Cell culture conditions (37°C, 5% CO2)
- Cell toxicity measured by lactate dehydrogenase enzyme released in the supernatant
- Not explicitly mentioned
- Not explicitly mentioned
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