Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

Meng, Fandan; Wu, Nai-Huei; Seitz, Maren; Herrler, Georg; Valentin-Weigand, Peter

Bronchial Epithelial Cell Growth Medium (BEGM), as previously described 57, 58 with some modifications, which is Bronchial Epithelial Cell basal Medium (BEBM, Lonza) supplemented with the required additives (Sigma). BEGM was refreshed at every 2 days intervals. Approximately after 4 days of cultivation cell monolayers reached 80% confluence and were subjected for further differentiation. Primary PTEC or PBEC were dissociated with trypsin (Life Technologies) and resuspended with Air-Liquid Interface medium (ALI medium), a mixture 1:1 of BEGM and Dulbecco's modified Eagle's medium (DMEM, Life Technologies) and 2.5 × 10 5 cells were seeded on type IV collagen pre-coated 0.33 cm 2 polyester transwell filters with polycarbonate membrane (0.4 μ m pore size, Corning Costar). Cells were then maintained at 37 °C in a humidified atmosphere of 5% CO 2 . After the cells reached confluence, ALI medium was removed from the apical compartment and cells were cultured under the ALI conditions, the PTEC or PBEC were cultured at least for additional 5 weeks for differentiation at 37 °C in a humidified atmosphere of 5% CO 2 . ALI medium in the basolateral compartment was renewed every 2 or 3 days and the apical surface was washed once per week with Hanks' balanced salt solution (HBSS, Life Technologies).