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

Samples were analyzed by using the inverse immunofluorescent microscope Nikon Eclipse Ti-S equipped with a 10x/0.30 and 40x/0.60 Plan Fluor objectives (Nikon). Subsequently, the area of the epithelial cells surface positive for green fluorescent bacteria was analyzed by applying the analySIS ® 3.2 software (Soft Imaging System) to quantify bacterial adherence. Three areas were chosen randomly for each sample and all experiments were repeated three times. Confocal immunofluorescence microscopy of samples were performed using a TCS SP5 confocal laser scanning microscope equipped with a 63x/1.30 NA glycerin HC PL APO objective and a 63x/1.40 oil HCX PL APO objective (Leica). Image stacks with a z or y-distance of 0.5 μ m per plane were acquired using 1 Airy unit pinhole diameter in sequential imaging mode to avoid bleed through. Maximum intensity projections were calculated for display purposes and adjusted for brightness and contrast using ImageJ/Fiji. FESEM studies of uninfected PBEC was performed as described previously 42 .