Nunc f96 microwell

The luciferin-luciferase assay (CellTiter-Glo^®2.0 Assay; Promega, Madison, WI, USA) was used to quantify the amounts of ATP released in the bathing solution of cultured cells in response to rinsing-induced mechanical stimulation^{27 (link)}. Briefly, 20 μl of bathing solution samples were individually placed in triplicate in white walled 96-well plates (Nunc F96 MicroWell; Thermo), and 100 μl CellTiter-Glo^®2.0 reagent was added directly to each well. Plates were incubated at room temperature for 15 minutes and were transferred to the Multilabel Plate Reader VICTOR X5 (PerkinElmer, Waltham, MA, USA), where luminescence was measured using a 1-sec integration time. The ATP concentration in the samples was calculated with the same procedure as for the in vivo study.

The luciferin‐luciferase assay (CellTiter‐Glo Luminescent Cell Viability Assay; Promega, Madison, WI) was used according to the manufacturer's protocol to quantify the amounts of ATP released in the bladder lumen and in the bathing solution of TRT‐HU1 cells. Briefly, 20 μL samples taken from the bladder of anesthetized mice and the bathing solution of TRT‐HU1 cells were individually placed in white‐walled 96‐well plates (Nunc F96 MicroWell; Thermo Fischer Scientific), and 100 μL CellTiter‐Glo reagent was added directly to each well. The plates were then incubated at room temperature for 15 min on a shaker, and transferred to the Multilabel Plate Reader VICTOR X5 (PerkinElmer, Waltham, MA), where luminescence was measured using a 1 sec integration time. The ATP concentration in the samples was calculated from standard curves constructed using ATP from 20 nmol L⁻¹ to 20 μmol L⁻¹.

rBap_B protein at 0.1 mg/ml was incubated in phosphate-citrate buffer pH 4.4. in a 96-well plate Nunc™ F96 MicroWell™ (ThermoFisher Scientific) with 200 µM of each polyphenol (Table 1). Before measuring thioflavin-T (Th-T) was added at the final concentration of 25 µM. Fluorescence emission spectra were recorded in the 460–600 nm range with an excitation wavelength of 445 nm using a 5 nm slit width for excitation and emission at 25 °C in the multi-mode reader Synergy H1 Hybrid Multi-Mode Reader.

Distension-induced urothelial ATP release into the bladder lumen was quantified from eight-week-old female C57BL/6 mice and from age-match female uCx43KO mice at both ZT8 (sleep/light phase) and ZT20 (active/dark phase). The experimental procedures were as previously reported [8 (link)]. Briefly, with animals under 2.0% isoflurane anesthesia, a 24 G catheter (SR-OT2419C, Terumo, Tokyo, Japan) was inserted into the urethra and held in place with a clamp (AM-1, Natsume Seisakusho Co. Ltd., Tokyo, Japan). The catheter was then connected to a tube filled with phosphate buffered saline (PBS) and a pressure reservoir. After bladder distention with 30 cm H₂O for 10 min, the PBS from the bladder lumen was collected and snap-frozen in liquid nitrogen. The luciferin-luciferase assay (CellTiter-Glo Luminescent Cell Viability Assay; Promega, Madison, WI, USA) was used to quantify the amounts of ATP released. Briefly, 20 μL of the collected PBS samples were individually placed in triplicate in white walled 96-well plates (Nunc F96 MicroWell; Thermo Fisher Scientific, Waltham, MA, USA), and 20 μL CellTiter-Glo^® 2.0 reagent was added directly to each well. Plates were incubated at room temperature for 10 min and then transferred to the Multilabel Plate Reader VICTOR X5 (PerkinElmer, Waltham, MA, USA), where luminescence was measured using a 1 s integration time.