Technical Reference #1859

1859.

Dysregulation of Dopamine Transporters via Dopamine D2 Autoreceptors Triggers Anomalous Dopamine Efflux Associated with Attention-Deficit Hyperactivity Disorder Erica Bowton; Christine Saunders; Kevin Erreger; Dhananjay Sakrikar; Heinrich J. Matthies; Namita Sen; Tammy Jessen; Roger J. Colbran; Marc G. Caron;8 Jonathan A. Javitch; Randy D. Blakely; and Aurelio Galli, Vanderbilt University, The Journal of Neuroscience, 30(1859), (2010)
Link To Paper

Materials & Methods:
Poly-D-lysine-coated glass bottom culture dishes (MatTek #P35GC-1.5-14-C) were coated with 10 g/ml laminin. A monolayer of rat C6 glial cells was plated 2–3 d before culturing (Rayport et al. 1992) neurons and maintained in Neurobasal-A media (Invitrogen #10888-022) supplemented with 10% fetal bovine serum 30 U/ml penicillin 30 mg/ml streptomycin and 0.6 mM L-glutamine. Midbrains from 1- to 4-d-old mouse pups were dissected cut into small pieces ( 1 mm3) and digested with papain solution consisting of 15 U/ml papain 1.25 mM cysteine 1.9 mM Ca2 100 U/ml DNase I 0.5 mM kynurenic acid. The papain digestion was performed for 5–20 min while stirring in the presence of 5% CO2 at 37°C. Cells were rinsed three times with neuronal media consisting of Neurobasal-A media supplemented with 10% fetal bovine serum (heatinactivated) 30 U/ml penicillin 30 mg/ml streptomycin 0.6 mM L-glutamine B-27 supplement and 0.5 mM kynurenic acid.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
HEK 293T