Fibronectin coated dishes

Anterior vaginal wall tissues were separated from the uterus of 8-week-old normal control female Sprague Dawley (SD) rats. At the anterior midline portion under sterile conditions, full-thickness tissues of the anterior vaginal wall (0.4 × 0.4 cm) were obtained from the vaginal cuff and the primary culture of rat smooth muscle cells was performed as previously described.³² (link) Tissues were cultivated in DMEM/F-12 (Invitrogen, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (FBS) (Gibico, Carlsbad, CA, USA), 1 mM glutamine (Invitrogen, Carlsbad, CA), 0.075% Na2HCO3, 100 U/mL penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA). The tissue was minced together with mechanically dissociated and PBS was simultaneously exploited to wash fragments of the tissue 3 times. Then they were plated onto fibronectin-coated dishes (Corning, NY, USA). After the initial outgrowth, clones with a morphology resembling the smooth muscle phenotype were patch cloned and propagated in a complete medium. The cultured rat vaginal SMCs were identified by immunocytochemical staining for alpha-smooth muscle actin (SMA) as described previously.³³ (link)