Exponentially growing drug-resistant cells were harvested and resuspended in PBS supplemented with 3% FBS. N-Flag-δ or N-Flag-α4 subunit expression at the cell surface was detected using an anti-Flag M2 (Mouse anti-DYKDDDDK IgG (Clone M2), which binds the Flag epitope in any position) conjugated to SureLight® APC, Columbia Biosciences, Frederic, MD). Cells were sorted at MGH Flow Cytometry Core facility using a BD 5 laser SORP FACS Vantage SE Diva system (BD Biosciences, San Jose, CA) using argon-ion laser excitation (633 nm, 35 mW). Emission for APC was detected at 650 nm. Dead cells and debris were excluded from the analysis by gating for intact cells using the forward and sideways scatter (488 nm, 320 mW). Uninduced cells were included as a negative control for autofluorescence. Data acquisition was carried out by analyzing 10 000 events/sample using CellQuest Software (BD Biosciences). FACS data were analyzed using FlowJo Software (Tree Star).
Following resorting of the cells in the pool inS1B Fig , one cell per well was seeded in a 96-well plate and cultured in antibiotic-enriched media. Some two dozen of these clones were analyzed for expression using flow cytometry and then by [3H]muscimol binding. The flow cytometry profile of the clone finally selected is shown in S1C & S1D Fig .
Following resorting of the cells in the pool in