Materials and methods Online
Mice and diet.
Mice were housed under standard conditions given free access to food and water. Experiments
were performed according to Dutch laws, approved by the Committee for Animal Welfare of
Maastricht University. Several experiments were performed. In the first experiment, 12-week
old female C57BL6 and ldlr-/- mice were fed a high fat diet containing 21% milk butter and
0.2% cholesterol (HFC), for 2, 4, 7 and 21 days. A single group was kept on a standard chow
diet until the age of 12 weeks and served as control group. In the second experiment, male
C57BL6, ldlr-/- and APOE2ki mice were put either on HFC for 7 days or were kept on chow
diet. In the third experiment, male and female C57BL6, ldlr-/-, and APOE2ki mice were fed
HFC or the high fat diet without the added cholesterol (HFnC) for 7 days. The HFnC diet
contained only the residual cholesterol derived from the butter component, i.e. 0.05%).
Collection of blood, sacrificing of the mice, and tissue isolation was done as described
previously (8).
Lipid analysis
Approximately 50 mg of frozen liver tissue was homogenized as described previously (8).
Both plasma and liver lipid levels were measured with enzymatic color tests (1489232,
cholesterol CHOD-PAP, Roche, Basel,Switzerland; TR0100, TG GPO-trinder, Sigma Aldrich,
St. Louis, MO, USA; 999-75406, NEFAC, ACS-ACOD, Wako Chemicals, Neuss, Germany) as
described before (8).
RNA isolation and first strand cDNA synthesis
Total RNA was isolated from approximately 25 mg of mouse liver tissues as described
previously (8). All applications were done according to manufacturers protocols. Total RNA
(500ng) from each individual mouse was converted into first strand cDNA with iScript cDNA
synthesis kit (170-8891, Bio-Rad, Hercules, CA, USA) according to the manufacturers
instructions.
Quantitative PCR
The changes in gene expression of inflammatory markers were determined by quantitative PCR
on a Bio-Rad MyIQ with the IQ5 v2 software (Bio-Rad, Hercules, CA, USA) by using IQ
SYBR Green Supermix with fluorescein (170-5006CUST, Bio-Rad, Hercules, CA, USA) and
10 ng of cDNA. For each gene a standard curve was generated with a serial dilution of a liver
cDNA pool. To standardize for the amount of cDNA, Cyclophillin A (Ppia) was used as the
reference gene. Primer sets for the selected genes were developed with Primer Express version
1.5 (Applied Biosystems, Foster City, CA, USA) using default settings. Primer sequences:
MCP1-forward, 5’-GCTGGAGAGCTACAAGAGGATCA-3’;
MCP1-reverse, 5’-ACAGACCTCTCTCTTGAGCTTGGT-3’;
CD68-forward, 5’-TGACCTGCTCTCTCTAAGGCTACA-3’;
CD68-reverse, 5-TCACGGTTGCAAGAGAAACATG -3’;
TNFa-forward, 5’-CATCTTCTCAAAATTCGAGTGACAA-3’;
TNFa-reverse, 5’-TGGGAGTAGACAAGGTACAACCC-3’;
Ppia-forward, 5’-TTCCTCCTTTCACAGAATTATTCCA-3’;
Ppia-reverse, 5’-CCGCCAGTGCCATTATGG-3’.
Data from qPCR was analyzed according to the relative standard curve method.
Taqman® Low Density Arrays
To perform gene-expression analysis on a medium scale, Taqman® Low Density Arrays
(TLDA) 96a (PN 4342259) were used. Each TLDA plate contained 4 x 96 annotated and
validated individual TaqMan® Gene Expression Assays (forward primer, reversed primer and
Taqman® Probe) (supplementary table 1). Per individual assay 2ng cDNA of a single liver was
loaded together with the TaqMan® Universal PCR Master Mix (PN 4324018). Of each group,
mRNA from 5 individual mice was used. TLDA plates were run on an ABI Prism 7900HT
Sequence Detection System with a TaqMan® Low Density Array Upgrade. Data was analyzed
by using RQ Manager 1.2 software. Technically failed assays were omitted from analysis.
Materials, equipment and software necessary to perform TLDA gene expression studies were
obtained from Applied Biosystems, Foster City, CA, USA. All data was normalized to Ppia
expression. Significant differences were determined with student t-tests; p-values < 0.05 were
considered significant.
Liver histology
4µm paraffin embedded liver sections were stained with Haematoxillin/Eosin (HE) and
Periodic acid Schiff (PAS)-diastase. Frozen liver sections (7µm) were fixed in acetone and
stained with CD68 (FA11) or Mac1 (M1/70). Pictures were taken with a Nikon digital camera
DMX1200 and ACT-1 v2.63 software (Nikon Corporation, Tokyo, Japan).
Electron microscopy.
Livers were freshly isolated from the mice and perfused and fixed overnight with 2.5%
glutaraldehyde (Ted Pella, Redding, CA, USA). Tissue fragments were washed and post fixed
in 1% osmium tetroxide. Tissues were subsequently dehydrated trough 100% ethanol, cleared
with propylene oxide, and embedded in epoxy resin. Sections of 1 μm were stained with
toluidine blue to identify the presence of foamy Kupffer cells. Next, sections of 70-90 nm were
cut on an ultra-microtome, mounted on Formvar-coated (1595E, Merck) 75 mesh copper grids
and counterstained with uranyl acetate and lead citrate before analysis on a Philips CM100
transmission electron microscope.
Statistical analysis
Data were analyzed using Graphpad Prism 4.0. Groups were compared using 2-tailed nonpaired t-tests or ANOVA with a Dunnet post test, based on the statistical relevance. Data is
expressed as means  SEM and considered significant at p  0.05.