genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
Pathogen Gene Expression Profiling
Bacterial control samples
Bacterial samples were taken as comparison and to control for additional experimental effects
(Fig. M.5.2 and Fig. M.5.3):
exponential growth phase at OD 0.4
staphylococci after 1 h, 2.5 h, and 6.5 h of incubation in the infection medium in 5 %
CO 2 -atmosphere at 37°C without agitation (i.e. in cell culture dishes in serum
containing medium but without presence of host cells)
non-adherent staphylococci after 1 h of co-incubation with eukaryotic cells and their
products in 5 % CO 2 -atmosphere and at 37°C
staphylococci after 2.5 h of anaerobic incubation in pMEM medium at 37°C
Cells were harvested using Killing Buffer (20 mM Tris pH 7.5, 5 mM MgCl 2 , 20 mM NaN 3 ) as
described above (see Material and Methods/Growth Media Comparison Study/Bacterial cell
harvest, page 55).
exponential growth phase
anaerobic
incubation:
2.5 h
infection
mix
serum control
with CO 2 exposure:
1 h, 2.5 h, 6.5 h
non-adherent staphylococci: 1 h
Fig. M.5.3:
Visualization of bacterial control samples in
the in vitro infection experiments for tiling
array analysis.
internalized staphylococci: 2.5 h, 6.5 h
S9 cells
staphylococci
Preparation of internalized staphylococci
The lysostaphin-containing medium was replaced by 1 ml Killing Buffer (20 mM Tris pH 7.5,
5 mM MgCl 2 , 20 mM NaN 3 ) with 150 mM NaCl. In this isotonic buffer the infected S9 cells were
scraped from the culture dish, resuspended and transferred to a 1.5-ml tube for further
processing while maintaining the integrity of the majority of host cells. All following steps were
performed on ice or at 4°C. Cells were pelleted (600 x g for 5 min) and fixed in ice-cold
acetone/ethanol (50 % v/v) for 4 min as described by Garzoni et al. (2007) followed by a
centrifugation step at 20000 x g for 3 min. The eukaryotic part of the cell pellet was lysed in RLT
buffer (Qiagen, Hilden, Germany) and homogenized twice using QIAshredder (Qiagen, Hilden,
Germany) and centrifugation at 20000 x g for 2 min. In this process staphylococci were not lysed
although they lost their viability. Therefore, the resulting pellet contained staphylococcal cells
and eukaryotic cell debris. Pellets from several plates processed in parallel were combined and
washed once with RLT buffer and four times with TE buffer (10 mM Tris/HCl pH 8, 1 mM EDTA
pH 8) to remove residual contaminations of the host cells. Staphylococcal cell pellets were flashfrozen
in liquid nitrogen and stored at −70°C until cell disruption.
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