5, and fractions of 1.5 mL were collected. Influence of proteinase K, sodium meta-periodate and dispersin B treatments on antigen integrity and Nutlin3a biofilm stability Overnight cultures of different S. epidermidis strains
in TSB were diluted 1:100 in 5 mL fresh TSB and incubated in 6-well flat-bottom tissue culture plates (Nunc) for additional 16–18 h at 37°C. Supernatants were removed and Selleckchem Wortmannin biofilms were detached using a cell scraper and suspended in 2 mL PBS. After brief vortex bacterial suspensions were adjusted to absorbance578 0.2. Aliquots of bacterial cultures (200 μL) were supplemented with 40 μL of 0.2 M sodium meta-periodate (Sigma), 2 μL of 100 μg/mL proteinase K (Promega, Madison, WI, USA), 2 μL of 1 mg/mL DspB and incubated at 4°C for 16 h, 37°C for 16 h and 37°C for 1 h and 5 h, respectively. Samples were applied onto immunofluorescence
slides at appropriate dilution and immunofluorescence tests performed as described above. For testing the stability of established biofilms, bacteria were grown overnight in 96-well cell tissue culture plates (Nunc) as described above. Medium was removed and PBS containing proteinase K (1 μg/mL) or DspB (10 μg/mL) or sodium meta-periodate (0.04 M) was added for 16 h at 37°C and at 4°C for sodium meta-periodate. Disruption of biofilm integrity was evaluated by assessment the absorbance at 570 nm. Absorption of antiserum 20-kDaPS and PIA antiserum AZD0156 were absorbed, as previously described , with slight modification. In brief, overnight cultures of selected strains were diluted 1:100 in TSB and incubated with shaking at 100 rpm for 18 h. Bacteria were harvested, washed two times in PBS and resuspended in PBS (absorbance578 =2). Aliquots of this bacterial preparation (50 μL) were Selleckchem 5-FU incubated with one μL of the respective antiserum diluted in 450 μL PBS overnight at 4°C on a rotating wheel. Bacterial cells were removed by centrifuging twice at 12,000 × g
for 15 min in a mini-centrifuge and the supernatants were filter sterilized. Antigen expression upon bacterial culture in chemically defined media S. epidermidis strains 1457, 1457-M10, and RP12 were subcultured daily for ten days in the following chemically defined broth media: RPMI1640, RPMI1640 + glutamine, IMDM, (Gibco, Invitrogen Life Science), TSB, TSB w/o dextrose and on blood agar plates. 20-kDaPS and PIA expression was assessed by immunofluorescence on day 1, 4, 7 and 10. Human monocyte derived macrophages Human peripheral blood mononuclear cells were isolated from buffy coats by density centrifugation on Ficoll density gradient (Biochrom AG, Berlin) and incubated for 2 h in RPMI-1640 medium supplemented with 10% heat-inactivated FCS (Biochrom AG, Berlin) and 2 mM L-Glutamine (HyClone) in 75 cm2 tissue culture flasks (Sarstedt Inc, Newton, NC, USA) at 37o C in a humidified, 5% CO2 atmosphere. Afterwards, non adherent cells were discarded and adherent cells were collected with a cell scraper.