Fluxor 2 green potassium ion channel assay

The selected clone stably
expressing Kv7.2/Kv7.3 channels was grown in DMEM supplemented with
10% FBS, 50 U/mL Pen-Strep, 2 mM l-glutamine (all purchased
from Gibco), and 4 μg/mL puromycin (Sigma) in a humidified atmosphere
at 37 °C with 5% CO₂. To perform the FluxOR II Green
Potassium Ion Channel Assay (Invitrogen), cells were seeded in a 96-well
Biocoat Poly-D-Lysine Cellware White/Clear Plate (Corning) at a density
of 1.6 × 10⁴ cells/well in 80 μL of medium.
24 h after seeding, the FluxOR assay was performed, following the
“Wash method” as described in the manufacturer’s
protocol. A kinetic dispense microplate reader (FLUOstar Optima) was
used to read the plate, setting the excitation filter at 485 nm and
the emission filter at 520 nm; “Stimulus buffer” prepared
as indicated in the protocol was automatically added to wells after
5 s of recording; the plate was read every 1 s, for 50 s. The results
were analyzed using Optima Data Analysis and Microsoft Excel.

To explore the effect of FPD on potassium channel opening, a FluxOR™ II Green Potassium Ion Channel Assay (Invitrogen, Paisley, United Kingdom) was carried out in VSMCs following the manufacturer’s guidelines. In brief, rat aortic smooth muscle cells were resuspended in the growth medium and were plated at 2 × 10³ cells per well in a 96-well plate. Cells were allowed to adhere overnight at 37°C. When the cells were attached, growth medium was replaced by the 1X loading buffer. Cells were incubated at 37°C for 60 min to facilitate dye entry. Further loading buffer was removed and assay buffer was added in each well. Treatment with 10 µM of FPD, 30 µM of FPD, 10 mM of TEA, and 80 mM of KCl were given for 20 min in different wells, and the FluxOR II assay was performed using Spectramax i3 (Molecular Devices, United States). The instrument was set at the excitation wavelength of 480 nm and the emission wavelength of 535 nm. Initially, 50 baseline readings were taken and after that 2 mM of thalium ion solution was injected and the fluorescence was studied for 5 min at every 0.4 s. The data were presented in the form of area under curve and a line diagram was plotted as relative fluorescence unit vs. time in seconds.

HL-1 cells were transiently incubated with miR-221/222 mimics for 24 h as described above. 48 h after start of transfection, cells were seeded onto a 96-well plate and incubated for another 24 h. FluxOR™ II Green Potassium Ion Channel Assay (Invitrogen™) was performed according to the manufacturer’s instructions using the Operetta CLS High-Content Analysis System (Perkin Elmer, Krakow, Poland) with a thallium ion (Tl⁺) concentration of 1 mM. To confirm that carbachol-induced (CCH, 10 µM final concentration, Sigma-Aldrich, Munich, Germany) thallium ion flux was indeed carried out by GIRK4, we performed initial experiments with tertiapin q (TQ, 100 nM final concentration, Alomone Labs, Jerusalem, Israel), an inhibitor of GIRK1/4. After obtaining the baseline fluorescence (F0), a buffer containing Tl⁺ alone (control) or additionally carbachol with or without tertiapin q was added and the fluorescence was measured every 15 s (30 time points after stimulation, Supplementary Figure S2).
Fluorescence data were normalized to baseline fluorescence (F/F0). The time course of F/F0 was integrated to obtain the area under the curve (AUC). Carbachol effect (CE) was calculated as AUC (carbachol)-AUC (control) for each individual experiment and afterward normalized to the mean CE of the corresponding mimic control (scramble).