After 2, 8.5 and 18.5 h of incubation the cell layers were washed thrice with PBS, detached by adding 500 μl trypsin solution (0.12% trypsin, 0.01% EDTA in PBS) per well (5 min, 37°C, 5% CO2, 90% humidity) and lysed for 5 min at 37°C with 0.025% Tween 20 to liberate the intracellular bacteria. Serial dilutions of the inoculum and the lysates were plated on blood agar plates to determine the number of colony forming units (cfu). Immuno-fluorescence For immuno-fluorescence staining ATM Kinase Inhibitor molecular weight an antibody against the C. diphtheriae surface proteome was used, which was raised in rabbits. For antibody generation, surface proteins were prepared as described [24].
As secondary antibodies Alexa-Fluor
488 (green) goat anti-rabbit IgGs and Alexa-Fluor 568 (red) goat anti-rabbit IgGs were used. Phalloidin Alexa-Fluor 647 was used for staining the cytoskeleton of D562 cells. All antibodies were diluted in blocking solution (2% goat serum, 2% BSA in PBS). Cell lines were seeded on round coverslips in 24 well plates 48 h prior to infection and fixed after the respective assay with 3% PFA in PBS (10 min at room temperature). For immuno-fluorescence staining the preparations were washed thrice with 1 × PBS and incubated with primary antibodies for at least 1 h at room temperature, washed thrice with PBS again, and subsequently EPZ-6438 clinical trial incubated with Alexa-Fluor 488 (green) goat anti-rabbit for 45 min. After permeabilization with 0.1% Triton X-100 (5 min room temperature) and three washing steps with PBS, staining with Alexa-Fluor 568 (red) goat anti-rabbit was carried out as described above. F-actin was stained in parallel with Phalloidin-Alexa-Fluor 647 (blue). Coverslips were mounted
on glass slides using Fluoroprep (Biomerieux, Craponne, France). Imaging was done on an AxioVert 200 M inverted optical microscope (Carl Zeiss Micromaging GmbH, Jena, Germany). Cobimetinib order Acknowledgements The authors wish to thank C. von Hunolstein (Istituto Superiore di Sanità, Rome) for providing strain ISS3319, ISS4060, ISS4746, and ISS4749, as well as H. Ton-That (University of Texas Health Science Center, Houston, TX) for SpaD antibodies, SpaD protein, and chromosomal DNA of strain NCTC13129. The help of R. G. Selleck AR-13324 Gerlach (Mikrobiologisches Institut des Universitätsklinikums Erlangen) to establish the epithelial resistance assay is gratefully acknowledged. This study was financially supported by the Deutsche Forschungsgemeinschaft for in frame of SFB 796 (projects B5, C1, and Z). References 1. Galazka A: The changing epidemiology of diphtheria in the vaccine era. J Infec Dis 2000,181(suppl 1):S2-S9.CrossRef 2. Hadfield TL, McEvoy P, Polotsky Y, Tzinserling A, Yakovlev AA: The pathology of diphtheria. J Infect Dis 2000,181(suppl 1):S116-S120.PubMedCrossRef 3.