The cells were allowed to adhere to the plate bottom


The cells were allowed to adhere to the plate bottom

for 45 min at 37 °C in a CO2 tissue culture incubator. FACS analysis of isolated cells Monoclonal FITC-labeled Antibodies were ordered from Miltenyi Biotec: anti CD14 clone TÜK4 and Immunotools (Friesoythe; Germany): GW-572016 anti CD11b-clone MEM-174. 1 μl anti CD14-FITC and 3 μl anti CD11b-FITC antibody were diluted in 50 μl of PBS, containing 0,5%BSA. 1 × 10e6 cells were added to each diluted antibody and were incubated for 30 min. at 4°C. After the incubation the cells were washed three times with 2 ml PBS/BSA by centrifugation for 5 min. at 400 g. Afterwards the cells were recovered in 0.5 ml of PBS/BSA and measured on a FACScalibure flow cytometer (BD, Heidelberg, Germany). The flow cytometer measurement revealed 12% CD14 and 28% CD11b positive cells in the mononuclear cell fraction after ficol gradient separation. The magnetic beads purified cells were enriched to 96% CD14+ and 98% CD11b+ respectively. Thus the magnetic bead separation produced a highly enriched monocyte fraction (Additional file 17, Figure S2). Bacterial cultures and

infection assay L. monocytogenes EGDe is a serotype 1/2a wild type isolate as described by Glaser P et al. 2001 [37]. S. aureus Gi.11268 and S. pneumoniae Gi.15342 are patient isolates characterized at the Institute of PF-3084014 cell line Medical Microbiology, Giessen. Overnight culture of L. monocytogenes EGDe and S. aureus Gi.11268 were grown in BHI medium at 37°C by continuous shaking. The Vorinostat ic50 over night cultures were diluted 1:50 and bacteria were grown in BHI medium reaching Phloretin an OD600 of 0.4 to 0.7. The number of viable bacteria was calculated using growth curves for both organisms. S. pneumoniae Gi.15342 was prepared by washing the bacteria with

prewarmed PBS from the surface of a Columbia-agar plate with an over night Streptococcus culture. The number of viable bacteria was calculated by using a dilutions curve at OD600. The required bacteria were collected by centrifugation at 5000 g for 10 min. and reconstituted in RPMI medium containing 1% FCS to a final concentration of 5 × 107 bacteria/100 μl. Adherent CD14+ cells were infected by adding 100 μl of the diluted bacteria suspension yielding a moi of 10. The tissue culture plaques were swung gently to mix the infectious medium and than centrifuged for 1 min at 900 g to ensure an even contact of the bacteria with the cells. 2 to 3 control wells received 100 μl of sterile medium. The cells were incubated for 1 h in a CO2 tissue culture incubator followed by cell lysis and RNA isolation. No antibiotics were used by the preparation of the cells and during the infection. RNA isolation For every bacterial pathogen and negative control the cells of at least two wells of a six well tissue culture plaque were lysed and total RNA was isolated. Prior to lysis culture medium was aspirated and cells lysed using RLT lysis buffer (Qiagen, Hilden, Germany).

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