Smooth muscle growth medium smgm

Either sex of ICR mice aged 4-7 days were sedated with diethyl ether and killed by cervical dislocation. Small intestine (from 1 cm below the pyloric ring to the cecum) and colon (from below cecum to rectum) were cut and pinned to the base of sylgard dish full with ice-cold Ca²⁺ free Hank’s solution (see solutions). Tissues were opened along the mesenteric border. After removing luminal contents, the mucosa and submucosa of tissues were peeled away by sharp dissection. Strips of intestinal or colonic muscle were equilibrated in Ca²⁺ free Hank’s solution for 30 minutes. The muscle strips were transferred into enzymic solution (see solutions) and incubated in a 37℃ water bath for 14 minutes. Then tissues were washed out 3 times with Ca²⁺ free solution and triturated with blunt pipettes to disperse cell lumps. Cells were plated on poly-L-lysine (200 μL; Sigma, St. Louis, MO, USA) coated sterile glass coverslips in 35 mm culture dishes and incubated at 37℃ in a 95% O₂-5% CO₂ incubator in smooth muscle growth medium (SMGM; Lonza, Walkersville, MD, USA) supplemented with 2% antibiotic-antimycotics (Gibco, Grand Island, NY, USA) and stem cell factor (5 ng/mL; Sigma,St. Louis, MO, USA). After 1 day incubation, the medium was replaced with SMGM without stem cell factor, then incubated further for 24 hours with the same conditions until performing the following experiments.