Sr ot2419c

After eight-week-old female C57BL/6 mice with or without Bmal1-knockout were anesthetized with 4% (induction) and 2% (maintenance) isoflurane, a 24 G catheter (SR-OT2419C, Terumo, Tokyo, Japan) was inserted into the urethra and clamped with a clip (AM-1, Natsume Seisakusho Co. Ltd., Tokyo, Japan), and connected to a tube filled with phosphate buffered saline (PBS). After bladder distention with 30 cm H₂O for 10 minutes, we collected the PBS from the bladder lumen. The ATP concentration in the samples was calculated from standard curves constructed using ATP from 20 nM to 20 μM.

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.