Asthmatic Airway Smooth Muscle Cell Culture Protocol

Human ASM cells (HASMC) were purchased from Lonza, Walkersville, MD. Donors were three adult asthmatic subjects (Catalog number 194850, Lonza Inc., Switzerland) and three disease-free and non-smokers (Catalog number CC-2576, Lonza Inc., Switzerland). HASMC were cultured in vitro as previously described [20] (link), [21] (link). Briefly, HASMC were cultured in growth medium supplemented with 10% FBS (Bio Whittaker), and maintained in an incubator containing 5% CO2 in air at 37°C. After attaining ∼95% confluence, the cells were starved in unsupplemented Ham's F12 media for 24 hr. For all experiments cells were used at passage 2–4. HASMC were stimulated with either TSLP (10 ng/ml R&D Systems, Minneapolis, MN, USA) or dsRNA analogue (Poly I:C 10 ug/ml, polyinosine-polycytidylic acid; InvivoGen San Diego, CA, USA). Concentrations of these reagents were based on prior studies [21] (link), [22] . After exposure to drugs and control media, confluent HASM cells were stimulated for 0, 24 or 48 h by adding dsRNA or TSLP to the wells. Cell supernatant was collected at the different time points and maintained at −80°C until further analysis.