genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
HOST CELL GENE EXPRESSION PATTERN IN AN
IN VITRO INFECTION MODEL
Host cell line and conditions of cell cultivation [performed by Melanie Gutjahr]
S9 cells were grown to confluency in 10-cm-diameter cell culture dishes with 10 ml eMEM cell
culture medium. Cell culture medium eMEM consists of 1x concentrated MEM (PromoCell GmbH,
Heidelberg, Germany) supplemented with additional 4 % FCS (fetal calf serum, Biochrom AG,
Berlin, Germany), 1 % non-essential amino acids (PAA Laboratories GmbH, Pasching, Austria), and
2 % L-glutamine (200 mM stock; PAA).
Bacterial growth medium and cultivation [performed by Melanie Gutjahr and Maren Depke]
Staphylococcus aureus RN1HG GFP (plasmid pMV158GFP) was grown in 100 ml pMEM
medium at 37°C with linear shaking of 125 strokes/min (stroke length 28 mm) in 500-ml-
Erlenmeyer bacterial culture flasks. Optical density (OD) was measured at 600 nm.
The adapted cell culture medium pMEM contains 1x concentrated MEM without sodium
bicarbonate (10x concentrate; Invitrogen, Karlsruhe, Germany), 1 % non-essential amino acids
(PAA Laboratories GmbH, Pasching, Austria) and 4 mM L-glutamine (PAA) and is supplemented
with 10 mM HEPES (PAA) and 2 mM of each L-alanine, L-leucine, L-isoleucine, L-valine, L-
aspartate, L-glutamate, L-serine, L-threonine, L-cysteine, L-proline, L-histidine, L-phenyl alanine,
and L-tryptophan (PromoCell GmbH, Heidelberg, Germany). The pH is adjusted to 7.4 with NaOH.
Use of this medium has been established by Sandra Scharf and has been first reported by
Schmidt et al. in 2010.
Cell culture infection model [performed by Melanie Gutjahr and Maren Depke]
When bacterial cultures reached an OD of 0.4 the S9 cells were infected with a multiplicity of
infection (MOI) of 25. Bacterial cultures and eukaryotic cell culture medium were mixed in a final
volume sufficient for inoculation of all cell culture plates processed in parallel. Subsequently, this
mix was added to the cell culture plates to ensure equal distribution of staphylococci on the host
cell layer and reproducible infection of all cell culture plates in each experiment.
6.36E+07 cfu/ml had been determined at OD 0.4 of S. aureus RN1HG GFP in pMEM (Melanie
Gutjahr). Confluent 10-cm-diameter cell culture plates contain 8.0E+06 S9 cells. Therefore, in
these experiments approximately 30 % of bacterial culture had to be included in the infection
medium to obtain a suspension of which 10 ml infect one cell culture plate with a MOI of 25
(3.14 ml bacterial culture in a total of 10 ml infection medium).
S9 cells and staphylococci were co-incubated for 1 h at 37°C in 5 % CO 2 -atmosphere.
Afterwards, the infection medium was replaced by eMEM containing 10 µg/ml lysostaphin (AMBI
PRODUCTS LLC, Lawrence, NY, USA) until harvesting of cells. Two time points were included in
this study: 2.5 h and 6.5 h after start of infection (Fig. M.4.1).
Control samples were treated accordingly. Only the volume of bacterial culture in the
infection mix was substituted by fresh, sterile bacterial cell culture medium pMEM.
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