Journal: ACS chemical neuroscience

Article Title: Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx

doi: 10.1021/acschemneuro.6b00301

Figure Lengend Snippet: Identification of TREK-2 bioactive lipid activators with a thallium flux assay. (A) Thallos fluorescence monitored before and after addition of Tl+ (arrow) to T-REx-TREK-2 cells induced with tetracycline (1 µg/mL, blue traces) or not induced (red traces). Inset is a Western blot run with T-REx-TREK-2 cell lysates with (+) or without (−) tetracycline induction and probed with a TREK-2 antibody. (B) Bioactive lipids that activate TREK-2 channels when preincubated with T-REx-TREK-2 cells for 5 min at a 10 µM concentration. (C) Tl+ flux (arrow) into thallos loaded TREK-2 expressing cells with (blue) or without (red) pretreatment with 11-deoxy prostaglandin F2α ± SEM (p < 0.001 after 300 s). (D) Dose–response curve for TREK-2 activation with 11-deoxy prostaglandin F2α ± S.E.M.; fitting determined an EC50 value of 0.294 µM. (E) Calculation of the Z′ using the slope of Tl+ influx for each well of a 384 well plate containing TREK-2 expressing cells treated with 11-deoxy prostaglandin F2α (top) or vehicle (bottom, Z′ of 0.752).

Article Snippet: Cells were loaded with 20 μL of Thallos Ion Channel reagent (AM) (TEFLabs, Austin, TX) dye loading solution for 45 min at ambient temperature for the HTS (dye solution: 50 μg of Thallos-AM in 20 μL of pure, dry DMSO with 10 μL of 20% pluronic F-127, diluted to 2 μM in assay buffer) and for 1 h for subsequent experiments.

Techniques: Flux Assay, Fluorescence, Western Blot, Concentration Assay, Expressing, Activation Assay

Journal: ACS chemical neuroscience

Article Title: Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx

doi: 10.1021/acschemneuro.6b00301

Figure Lengend Snippet: Optimization of the secondary TREK-1 Tl+ flux assay. (A) Thallos fluorescence (F488) monitored before and after addition of Tl+ (arrow) to T-REx-TREK-1 cells induced with tetracycline (1 µg/mL, blue) or not induced (red) ± SEM. (B) Tl+ flux (arrow) into thallos loaded T-REx-TREK-1 expressing cells with (red) or without (blue) 10 min pretreatment with 1.8 µM 11-deoxy prostaglandin F2α ± SEM.

Article Snippet: Cells were loaded with 20 μL of Thallos Ion Channel reagent (AM) (TEFLabs, Austin, TX) dye loading solution for 45 min at ambient temperature for the HTS (dye solution: 50 μg of Thallos-AM in 20 μL of pure, dry DMSO with 10 μL of 20% pluronic F-127, diluted to 2 μM in assay buffer) and for 1 h for subsequent experiments.

Techniques: Flux Assay, Fluorescence, Expressing