Female rat PASMC was isolated as described previously.23 (link) In brief, the whole lungs or the left lobe was excised from the rat and immediately placed in cold DMEM (Dulbecco’s Modified Eagle Medium) without glutamine and pyruvate (catalog No. 11960-044; Gibco). Under the hood, clean vessels from the lungs were cut and transferred to 2 mL of 1 mg/mL collagenase-2 (catalog No. LS004176; Worthington) in DMEM without glutamine and pyruvate for 20 minutes at 37 °C. The supernatant was aspirated carefully, and 10 mL of warm DMEM with glutamine supplemented (catalog No. 11965-092; Gibco) with 20% fetal bovine serum (catalog No. 25-5144; Geneclone) was added. This was then plated in a 10-cm dish covered with gelatin (catalog No. ES-006-B; Millipore). To ensure adequate cell number, cells were pooled from 5 different rats. Every 2 to 3 days, half of the media was changed to DMEM with glutamine. After 2 to 3 passages, the cells were stained for smooth muscle actin (anti-α-SM actin [1A4], Alexa-Fluor488 ab184675, Abcam, 1:100, isotype IgG2a) using FACS performed in a Novocyte 2000 (ACEA Biosciences) instrument.