The IonFlux system uses well-plate microfluidic devices called IonFlux Plates. These are SBS-standard well plates with a network of micron-scale channels that run underneath and in between wells of the plate. The network consists of a repeated pattern (Figure 2) interfacing to 12 wells on the plate. This pattern is repeated eight times on 96-well plates (IonFlux 16) and 32 times on 384-well plates (IonFlux HT).
Each pattern is an independent experimental unit that obtains recordings from two cell ensembles in parallel. Both ensembles in each pattern are exposed to eight independent compounds or a concentration series of a single compound.
Cells expressing GABAA (α1/β3/γ2-HEK, Millipore PrecisION) were cultured in 225 cm2 filter-top flasks containing DMEM/F12 glutamax, G-418, Hygromycin B, and Puromycin at 37°C and 5% CO2. The cells were kept below 90% confluency. For cell isolation, flasks were first washed with 2 mL of Ca- and Mg-free PBS, followed by Detachin solution after which cells were given two to five minutes of further Detachin treatment.
After release, the cell suspension was spun for 90 seconds at 1,000 rpm prior to being resuspended in extracellular solution (mM): 137 NaCl, 4 KCl, 1 MgCl21, 1.8 CaCl2, 10 HEPES, 9 glucose, pH 7.4 with NaOH 10. The intracellular solution for the whole-cell voltage clamp contained (mM): 130 K Aspartate, 5 MgCl2, 5 EGTA, 4 Tris-ATP, 10 HEPES, pH 7.2 with KOH. To evoke inward Cl- currents, cells were trapped in ensembles of 30 cells each. Whole-cell voltage clamp was established by applying suction, and GABA was applied while holding the membrane at -80 mV.
Assay validation was performed by first running a detailed analysis of the response to increasing doses of GABA, in order to determine the EC50 and EC20 concentrations, which were 3.4 µm (literature EC50 value 6 µM) and 1 µM, respectively. Next, we performed concentration-response measurements for known GABA antagonists: Picrotoxin showed an IC50 of 4.6 µM (literature IC50=2.4 µM), bicuculine IC50=15 µM (literature IC50=2 µM) and gabazine IC50=7.2 µM (literature IC50=6.6 µM).