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CHO cells stably expressing cloned rat µ- or d-opioid receptors were maintained in Ham's F-12 medium containing 10% fatal calf serum and G418 (Life Technologies; 700 µg/ml). Confluent cells were detached with a PBS solution containing 0.05% trypsin and 0.53 mM EDTA, harvested, and after diluting 1:10 cells were plated on circular glass microscope coverslips in 35 x 10-mm culture dishes. Cells were grown in the incubator for 24 to 48 h before fluorescent-peptide ligand receptor binding.

NG-108-15 cells were maintained in Ham's F-12 medium plus 10% heart extract and was cultured in monolayers on the circular coverslips 2 to 3 days before fluorescent binding experiments. SH-SY5Y cells were maintained in F-12/Dulbecco's minimal essential medium (1:1) medium plus 10% fatal calf serum and then were plated and allowed to grow on the coverslips overnight before the experiments.

All fluorescent-peptide ligand receptor binding and internalization experiments were observed concurrently during incubation while cells were vial. Before binding experiments cells were washed twice and left in Ham's F-12 medium plus 10% fetal calf serum at room temperature and then observed their autofluorescence to establish an initial emission background level. The medium was then replaced with a solution of fluorescent ligand in Ham's F-12 medium containing bestatin (10 µM), thiorphan (1 µM), and 10% fatal calf serum. Cells were incubated for various times at 4°C for binding study and 35°C for internalization study. Incubation was terminated by removing the medium and washing the cells 5 to 10 times with Ham's F-12 medium plus 10% fetal calf serum. To determine nonspecific binding of the fluorescent peptide ligand, cells were pretreated with naloxone (10 µM) and subsequently incubated in a solution of fluorescent ligand plus naloxone (10 µM).

Arttamangkul S. et al. Binding and internalization of fluorescent opioid peptide conjugates in living cells. Mol Pharmacol 58, 1570-1580 (2000)