Pico145 (HC-608)

Cell experiment:

Cells are seeded at 90% confluence into 96-well clear-bottomed poly-d-lysine-coated black plates for HEK 293 cells and clear-bottomed Nunc plates for A498 cells, Hs578T cells, and HUVECs 24 h before experimentation. Fura-2 Ca2+ indicator dye is used to monitor changes in intracellular ionized Ca2+ concentration. To perform the experiment, the cells are incubated for 1 h with fura-2-AM (2 μM) in standard bath solution (SBS) at 37°C in the presence of 0.01% pluronic acid. SBS contains 135 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 8 mM glucose, and 10 mM Hepes (pH titrated to 7.4 using NaOH). Subsequently, the cells are washed twice with SBS before adding Pico145 or ML204 for 30 min before making Ca2+ measurements. The fura-2 fluorescence is recorded using a 96-well fluorescence plate reader and the excitation wavelengths of 340 and 380 nm. For TRPV4 recordings, fluo-4/AM is used in place of fura-2/AM, and 2.5 mM probenecid is included to inhibit leak of fluo-4. Fluo-4 is excited at 485 nm, and emitted light is collected at 525 nm. Ca2+ is indicated by the ratio of the fluorescence (F) emission intensities for the two excitation wavelengths. Measurements are made at room temperature (21 ± 3°C)[1].

References:

[1]. Rubaiy HN, et al. Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels. J Biol Chem. 2017 May 19;292(20):8158-8173.