Multiplex assay

For glucose tolerance tests (GTT), ApoE^−/− mice transplanted with WT and C/EBPβ^−/− bone marrow cells and fed LF and HF/HC diets were fasted for 4 h and injected intraperitoneally with glucose (2 mg/g body weight), as described previously [11 (link),13 (link)]. Blood samples were collected from the tail vein at 0, 30, 60, 90 or 120 min and glucose was measured using a glucometer. Area under the curve (AUC) of glucose was calculated as described previously [14 (link)]. To circumvent influences of glucose injection on hepatic gene expression, animals were sacrificed one week after the last GTT, i.e. after 11 weeks on the experimental diets. Serum insulin and adiponectin levels were measured by ELISA kits from ALPCO (Windham, NH). Serum cytokines (IL-1β, TNFα, MCP1, and IL-6) was measured by multiplex assay (Bio-Rad, Hercules, CA). Serum total cholesterol, triglycerides, HDL cholesterol, and non-HDL cholesterol were analyzed enzymatically using a Beckman Coulter AU automated chemistry analyzer (Beckman Coulter, Brea, CA).

Spleens taken aseptically from euthanized mice (4 mice/group) were dissociated individually. The splenic cell suspensions were depleted of red blood cells (RBC) using RBC lysis buffer (Sigma) and splenocytes were washed extensively with cold PBS. T cells were purified from splenocytes by MagniSort™ Mouse T-cell Enrichment Kit according to instruction (eBiosciences, CA). Purity was routinely more than 90%, as assessed by flow cytometry using monoclonal antibodies specific for mouse CD4, CD8, and CD3 cells. Cells resuspended in RPMI 1640+GlutamaxTM (Gibco) supplemented with 5% fetal bovine serum and 100 μg/ml penicillin/streptomycin were seeded in 96 well plates (1×10⁶/well) and stimulated with either YpL (4 μg/ml), the F1 antigen (4 μg/ml) or PMA (20 ng/ml) plus ionomycin (1μg/ml) as a positive control. After 72 h, the supernatants of cell cultures were collected for cytokine analysis using multiplex assay (Bio-Rad). The cells stained with CD4-phycoerythrin (PE) (clone RM4-5), CD8-allophycocyanin (APC) (clone 53-6.7) and then intracelullarly stained with IFN-γ-fluorescein isothiocyanate (FITC) (clone XMG1.2) were analyzed by Flow cytometry as mentioned in a previous report [35 (link)]. Data was collected on a Beckman Coulter FC500 and analyzed using FCS Express 4 software.

Whole lung homogenates in PBS were centrifuged and supernatants analyzed for pro-inflammatory cytokines. IL-12p40, IL-12p70 and TNF-α were measured by ELISA (eBioscience, San Diego, CA and R&D Systems, Minneapolis, MN) and CCL2, CCL3, CCL4, CXCL1 were detected by multiplex assay (Bio-Rad, Hercules, CA). Bronchoalveolar lavage (BAL) was performed using 0.5 ml PBS aliquots. BAL fluid was spun for 15 minutes at 1500 g, and the supernatants were analyzed.