反应详情

温度

37 °C

[3H]DA uptake was measured in cultures at day 7, and all the solutions were maintained at 37° C. The growth medium was removed, and the cultures were rinsed twice with 0.25 ml of uptake buffer, which consists of HBSS (Gibco 11201-092) supplemented with 28 mM glucose, 15 mM HEPES, 1 mM ascorbic acid (an antioxidant), and 0.5 nM pargyline (a monoamine oxidase inhibitor). The cultures were then incubated with 0.25 ml of fresh uptake buffer containing 50 nM [3H]DA (NEN/DuPont) for 20 min at 37° C. [3H]DA uptake was stopped by removing the incubation mixture, and cells were then washed twice with 0.5 ml of the uptake buffer at room temperature. To release [3H]DA from the cells, the cultures were lysed with 0.2 ml of 0.1 N NaOH for 1 h at room temperature, the lysate was then added to 1 ml of Microscint-20 (Packard 6013621), and counted for radioactivity in a microplate scintillation counter (Packard TopCount, NXT). Background values were obtained by adding to the uptake buffer 0.5 mM GBR-12909, a specific inhibitor of the high-affinity uptake pump of the dopaminergic neurons, and were usually <5% of the 3H uptake in untreated control cultures. Forskolin (Fk, 25 μM) was employed as a positive control.

1. wait[3H]DA uptake was measured in cultures at day
2. customThe growth medium was removed
3. washthe cultures were rinsed twice with 0.25 ml of uptake buffer
4. additionbuffer containing 50 nM [3H]DA (NEN/DuPont) for 20 min at 37° C. [3H]DA uptake
5. customby removing the incubation mixture, and cells
6. washwere then washed twice with 0.5 ml of the uptake buffer at room temperature
7. additionthe lysate was then added to 1 ml of Microscint-20 (Packard 6013621)
8. customBackground values were obtained