Primary smooth muscle cell isolation and culture

All procedures were carried out in a laminar flow hood using sterile techniques. Mouse urinary bladder was placed in a Petri dish (6 cm diameter) containing DMEM supplemented with antibiotics. The adjacent connective and fatty tissues were removed. Urinary bladder was opened longitudinally and bladder neck including trigone and urethra were resected. The detrusor smooth muscle was cut into tiny pieces using surgical scissors. Tissue fragments were washed five times with PBS and digested in 1% collagenase (Gibco, Carlsbad, USA) at 37 °C for 30 min on a shaker. After digestion, enzymes were neutralized by addition of an equal volume of DMEM medium containing 10% FBS. Resulting suspensions were filtered through 100 μm nylon cell strainers (BD, USA) and centrifuged at 180 × g for 5 min. The cell pellet was resuspended in culture medium, and cells were cultured at 37 °C in 5% CO₂ and 95% humidity. Growth medium was changed every 2–3 days. Human bladder primary smooth muscle cells (Catalog: FC-0043) were purchased from LifeLine Cell Technology (Frederick, MD) and were cultured in VascuLife SMC culture medium (Catalog: LL-0014) with 5% FBS, 10 mM l-glutamine, 30 µg ml⁻¹ ascorbic acid, 5 ng ml⁻¹ recombinant human EGF and FGF basic at 37 °C in 5% CO₂ and 95% humidity. We used primary cultured mouse bladder smooth muscle cells (1st passage) and primary cultured human smooth muscle cells (3rd–4th passage) for Ca²⁺ imaging and for detection of mRNA and protein. The functional contractile phenotype of these primary cultured smooth muscle cells was confirmed before each experiment.