Membrane potential blue kit

The methods were as described previously.^{7 (link)} In brief, fluorescent membrane potential dye
(Membrane Potential Blue kit, Molecular Devices) was diluted in Flex
buffer (10 mM HEPES, 115 mM NaCl, 1 mM KCl, 1 mM CaCl₂,
1 mM MgCl₂, and 10 mM glucose, pH 7.4) and added to each
well. Following incubation at 37 °C for 30 min, fluorescence
was measured in a FlexStation 3 (Molecular Devices) at 2 s intervals
for 200 s. 5-HT (Sigma) was added to each well after 20 s. Data were
normalized to the maximum ΔF and analyzed using
Prism (GraphPad Software Inc.).

These methods were as described previously [13 (link)]. In brief, fluorescent membrane potential dye (Membrane Potential Blue kit, Molecular Devices) was diluted in Flex buffer (10 mM HEPES, 115 mM NaCl, 1 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM glucose, pH 7.4) and added to each well. The cells were incubated at 37 °C for 45 min and fluorescence measured in a FlexStation 3 (Molecular Devices) at 2 s intervals for 400 s. GABA (Merck) was added to each well after 20 s. Peak fluorescence (F) at each [GABA] was normalised to the maximum ΔF, and data were analysed using Prism (v6, GraphPad Software Inc., San Diego, CA, USA), fitting concentration–response data to the four-parameter logistic equation: $F=Fmin+\frac{\left(Fmax-Fmin\right)}{1+{10}^{(\log \left(E{C}_{50}\left[L\right]\right)n_H}}$ , where [L] is the ligand concentration, n_H is the Hill coefficient, and Fmax and Fmin are the maximal and minimal fluorescence levels for each dataset (NB n_H values are reported but not discussed as it is difficult to meaningfully interpret these values when using an indirect assay). Statistical analysis was performed using ANOVA with a Dunnett’s multiple comparisons post test.

As described previously (Price and Lummis, 2005 (link)) cells were incubated for 45 min with fluorescent membrane potential-sensitive dye (Membrane Potential Blue kit, Molecular Devices) diluted in Flex buffer (10 mM HEPES, 115 mM NaCl, 1 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM glucose, pH 7.4), and subsequently assayed at room temperature for 180 s, with readings every 2 s. 5-HT was added to each well after 20 s. Concentration-response curves were generated by iterative fitting in GraphPad Prism 7 (after normalization to max ΔF) with the equation $y=a+\frac{b-a}{1+{10}^{({\text{n}}_{\text{H}}({\text{logEC}}_{{50}^{\text{-x}}}))}}$ where y is the fluorescent response, x is log[5-HT] (log of the concentration of ligand), a is the minimum response, b is the maximum response, and n_H is the Hill slope.