5 μl of each sample were fixed on a glass slide by drying using compressed air. An AFM instrument (MFP-3D, Asylum Research, Santa Barbara, CA) with Selleck SCH772984 standard silicon cantilever probes (NCH-W, Nanosensors, Neuchatel, Switzerland) was used under ABT-263 mouse ambient laboratory conditions and operated in tapping mode [26]. Measurement of transepithelial resistance D562 cells were seeded in transwells (6.5 mm, 0.4 μm, polyester membrane, 24 well plate, Corning Costar) at a density of 5 × 104 cells per well and

cultivated in DMEM (Dulbecco’s modified Eagle’s medium, PAA; high glucose, 10% FCS, 2 mM glutamine) for 14 days until they build a transepithelial resistance of at least 1600 Ω·cm-2. Bacteria were subcultured (OD600 of 0.1 from overnight cultures) in 20 ml HI broth for 3.5 h. The pellet was resuspended in 500 μl 1 × PBS. 50 μl of the suspension were used for infection. Measurements of transepithelial resistance of D562 cells during the JPH203 clinical trial infection with C. diphtheriae were carried out with a volt-ohm-meter (EVOM2, World Precision Instruments, Berlin, Germany) every 30 min. After 3 h the supernatant of infected

D562 cells was removed and the cells were incubated in fresh DMEM overnight to avoid detrimental effects of excessive bacterial growth. Adhesion assays D562 cells were seeded in 24 well plates (bio-one Cellstar, Greiner, Frickenhausen, Germany) at a density of 2 × 105 cells per well 48 h prior to infection. Bacteria were subcultured (OD600 of 0.1 from overnight cultures) in HI broth for 3.5 h and adjusted to an OD600 of 0.2. A master mix of the inoculum was prepared in DMEM without penicillin/streptomycin at a MOI of 200 (viable counts experiments). The plates were centrifuged for 5 min at 500 × g to synchronize infection and subsequently incubated for 1.5 h. The cells were washed with PBS nine

times, detached with 500 μl trypsin solution (0.12% trypsin, 0.01% EDTA in PBS) per well (5 min, 37°C, 5% CO2, 90% humidity) and lysed with 0.025% Tween 20 for 5 min at 37°C. Serial dilutions were made in pre-chilled 1 Cytidine deaminase × PBS and plated on blood agar plates to determine the number of colony forming units (cfu). From this, the percentage of invasive bacteria was calculated [24]. Epithelial cell invasion model D562 cells were seeded in 24 well plates (bio-one Cellstar, Greiner, Frickenhausen, Germany) at a density of 2 × 105 cells per well 48 h prior to infection. Overnight cultures of C. diphtheriae grown in HI were re-inoculated to an OD600 of 0.1 in fresh medium and grown aerobically for another 3.5 h. An inoculum of approximately 1.6 × 108 bacteria ml-1 (MOI = 200) was prepared in DMEM without penicillin/streptomycin and 500 μl per well were used to infect the D562 cells. The plates were centrifuged for 5 min at 500 × g to synchronize infection and subsequently incubated for 1.5 h (37°C, 5% CO2, 90% humidity).