TRACT assays (xCELLigence)

Label-free whole-cell TRACT assays were performed using the xCELLigence real-time cell analyzer (RTCA) system as described in previous publications^22,32. In principle, xCELLigence RTCA measures the impedance that is generated by cells that adhere to the gold-coated electrodes and cover the bottom of microtiter E-plates. Any change in adhesion, cell number, proliferation rate and morphology (e.g., as a result of pharmacological perturbations) is measured as an increase or decrease of impedance over time. Impedance values, which are measured continuously at a frequency of 10 kHz, for each well are converted by the RTCA software to the dimensionless parameter Cell Index (CI) using the following formula:

where Z_i is the impedance at any given time point and Z₀ is the baseline impedance that is measured at the start of each experiment¹⁶.

All assays were performed at 37 °C and 5% CO₂ in 96-well E-plates in a total volume of 100 µl per well. Depending on the amount of compound additions during an experiment, background impedance at the start of each experiment was measured in 45 µl (1 addition) or 40 µl (2 additions) culture medium. Cells were seeded manually in the wells in a volume of 50 µl. Compounds were added in 5 µl per addition using a VIAFLO 96 handheld electronic 96 channel pipette (INTEGRA Biosciences, Tokyo, Japan).

To demonstrate reproducibility of the TRACT experiments, cells were used from at least two different cell batches. In addition, cells were used at different passage numbers, ranging from p2 to p11. U2OS-mock or U2OS-DAT cells grown to 50–70% confluence were detached 24 h post-transfection from 10 cm plates with PBS/EDTA. Background impedance in 96-well E-plates was measured using culture medium containing a final concentration of 5 mM sodium butyrate. Subsequently, cells were seeded at 40,000 cells/well in culture medium. The E-plate was left at room temperature for 30 min and placed in the recording station. Impedance was measured overnight every 15 min. Cells were treated 17–19 h after seeding based on previous reports^12,22.

JumpIn-DAT cells grown to 70–80% confluence were briefly trypsinized from 10 cm plates prior to use in the assay. Baseline impedance was measured using culture medium containing dox (1 pg/ml–1 µg/ml) or vehicle (milliQ water). Subsequently, cells were seeded at 60,000 cells/well in culture medium. The E-plate was left at room temperature for 30 min and placed in the recording station. Impedance was measured overnight every 15 min. Cells were treated 22–24 h after seeding as dox-induced protein expression is optimal after 24 h according to the JumpIn cells manufacturer’s protocol (Thermo Fisher Scientific)³³.

In antagonist experiments, cells were pretreated by the addition of a GPCR antagonist (1 µM; SCH23390, raclopride, doxazosin, yohimbine, propranolol). In TRACT assays, cells were pretreated with a DAT inhibitor (10 µM or increasing concentrations (100 pM–10 µM); GBR12909, cocaine) or a vehicle control (0.1% DMSO in PBS). Final amounts of DMSO in each well were kept at 0.1%. Impedance was measured every minute after the addition for 60 min.

Cells were stimulated by the addition of dopamine (concentration depending on type of assay) or a vehicle control (1 mM ascorbic acid in PBS). Note, ascorbic acid was used in the presence of dopamine to prevent its oxidation in culture medium. In antagonist experiments, cells were stimulated with a submaximal (EC₈₀) concentration of dopamine. In TRACT assays to determine the inhibitory potency of DAT inhibitors, cells were stimulated with a submaximal (EC₂₀) concentration of dopamine. Impedance was measured initially every 15 s after the addition for 25 min, then every minute for 10 min, every 5 min for 50 min and finally every 15 min. For U2OS-mock and U2OS-DAT cells, impedance was measured for 120 min after stimulation. For JumpIn-DAT cells, impedance was measured for 30 min after stimulation.