genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
GENE EXPRESSION PATTERN OF BONE-MARROW DERIVED
MACROPHAGES AFTER INTERFERON-GAMMA TREATMENT
Stem cell preparation, cultivation, and differentiation to macrophages [performed by
Katrin Breitbach]
Preparation and cultivation of mouse stem cells and their differentiation into bone-marrow
derived macrophages (BMM) was conducted as described by Eske et al. in 2009. Briefly, bone
marrow cells from tibias and femurs of n = 3 or n = 15 BALB/c or n = 4 or n = 15 C57BL/6 mice
were prepared in sterile conditions, pooled and cultivated for ten days using the serum-free
BMM-medium supplemented with GM-CSF as established by Eske et al. (2009). Differentiated
BMM were harvested on day 10 for consecutive experiments.
Interferon-γ activation of bone marrow derived macrophages [performed by Katrin Breitbach]
Mature BMM were seeded in 6-well-plates with 0.8E+06 to 1.5E+06 cells / well. BMM in half of
the wells were treated for 24 hours by addition of 300 units/ml IFN-γ (Roche, Mannheim,
Germany) in serum-free BMM-medium, the other half was cultivated for the same time in the
same medium without IFN-γ. For transcriptome analysis, 1.6E+06 to 4.5E+06 cells / sample were
available, while proteome analysis needed a higher cell number and therefore had a sample size
of 1.0E+07 to 1.5E+07 cells.
Sample [Katrin Breitbach] and RNA preparation [Maren Depke] for transcriptome analysis
Medium was carefully removed from the sample wells, and BMM were lyzed in 1 ml
TriReagent per sample (Sigma, Steinheim, Germany) by repetitive pipetting. After incubation at
room temperature for 15 min, the lysate was flash-frozen in liquid nitrogen and stored at −70°C
until RNA-preparation.
RNA-preparation took place using a combined protocol of phenol-based preparation and
column-based purification. Chloroform was added to TriReagent lysates, samples were vigorously
shaken and incubated at room temperature for 5 min. Organic and aqueous phase were
separated by centrifugation for 15 min at 12000 x g and 4°C. Afterwards, the aqueous
supernatant was mixed with 0.5 ml isopropanol (2-propanol) and transferred to RNeasy Mini
columns (Qiagen GmbH, Hilden, Germany). The following steps of RNA purification were carried
out according to the manufacturer’s instructions including the optional DNase-treatment (RNasefree
DNase Set, Qiagen GmbH, Hilden, Germany). After ethanol precipitation, the RNA was
quantified spectrophotometrically, and its quality was verified using an Agilent 2100 Bioanalyzer
and RNA Nano Chips (Agilent Technologies Inc., Santa Clara, CA, USA).
Affymetrix DNA array analysis
Four strain-treatment combinations were included into this study: 1) medium control BALB/c
BMM, 2) IFN-γ treated BALB/c BMM, 3) medium control C57BL/6 BMM, and 4) IFN-γ treated
C57BL/6 BMM. Three biological replicates were analyzed for each of the four sample groups
described before.
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