Ez cytox cell viability kit

The toxicity of S. pneumoniae in the presence or absence of ASD was detected by evaluating HMEEC viability upon treatment with S. pneumoniae or ASD, or co-treatment with both. HMEECs were kindly provided by Dr. Lim (House Ear Institutes, Los Angeles, CA, United States) (Chun et al., 2002 (link)). HMEECs were cultured (1 × 10⁴) in a 96-well plate in airway epithelial cell growth medium (PromoCell GmbH, Sickingenstr Heidelberg Germany) supplemented with bovine pituitary extract (0.004 ml/ml), epidermal growth factor (10 ng/ml), insulin (5 μg/ml), hydrocortisone (0.5 μg/ml), epinephrine (0.5 μg/ml), triiodo-L-thyronine (6.7 ng/ml), transferrin (10 μg/ml), retinoic acid (0.1 ng/ml), and 1% fetal bovine serum for 24 h at 37°C in 5% CO₂. Then, cells were exposed to ASD (300 μg/ml) in serum-free medium for 8 h, followed by S. pneumoniae treatment (MOI 10) for 15 h. The viability of HMEECs was determined by using EZ-cytox cell viability kit (Dogenbio, South Korea) as per the manufacturer’s instruction, and absorbance was measured at 450 nm.

The HMEECs were maintained and routinely cultured in airway epithelial cell growth medium (PromoCell GmbH, Sickingenstr Heidelberg, Germany) supplemented with bovine pituitary extract (0.004 mL/mL), epidermal growth factor (10 ng/mL), insulin (5 µg/mL), hydrocortisone (0.5 µg/mL), epinephrine (0.5 µg/mL), triiodo-L-thyronine (6.7 ng/mL), transferrin (10 µg/mL), retinoic Acid (0.1 ng/mL) and 1% fetal bovine serum and 5% CO₂. In a 96-well plate, 1 × 10⁴ HMEECs were seeded and incubated at 37 °C with 5% CO₂ for 24 hr. Following incubation, cells were washed with PBS and medium was replaced with UP (200 µg/mL) containing medium (antibiotic free medium) and further incubated for 8 hr. The HMEECs pre-exposed to UP were infected with S. pneumoniae (MOI 10) for 15 hr. The HMEECs viability was detected using the EZ-cytox cell viability kit (Dogenbio, Korea) as per manufacturer’s instruction.

The silica powder and plate were obtained from Merck (Darmstadt, Germany). The Sephadex LH-20 resin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The EZ-Cytox cell viability kit was obtained from DoGenBio (Seoul, Korea). The other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).