Enzymatic kit

Aliquots of the perfusate were collected at 1-min intervals, and LDH activity was measured by an enzymatic kit (Sigma-Aldrich) sensitive to detect changes in the µU/mL range, and expressed as U/L. Similar determinations were carried out in isolated cardiac myocytes and H9C2 cells. In both cell models, LDH activity was assessed as a percentage of the total amount of the enzyme released from digitonin-permeabilized cells.
Protein content was determined by Lowry assay (Lowry, Rosebrough, Farr, & Randall, 1951 (link)), using bovine serum albumin as a standard.
After excision of the heart, blood samples were collected by heparinized capillaries. The tubes were centrifuged at 500 × g for 20 min, and plasma alcohol content was determined by enzymatic assay (Sigma-Aldrich). Exposure to Lieber-DeCarli diet for 3 weeks resulted in a plasma ethanol concentration of 42 ± 3 mmol/L (n = 8), which is consistent with other published reports (Fisher et al., 2001).

Glucose (1.5–2.0gkg⁻¹ after 15 h fast unless otherwise indicated in figure legends) and insulin (1Ukg⁻¹ after 5 h fast) tolerance test were performed on C57BL/6J mice 3–4 or 9–10 weeks after initiation of HFD. For experiments with Ccr2⁻⁻ mice, 4-week-old WT and Ccr2⁻⁻ mice were acclimated to 30°C for 2 weeks prior to initiation of HFD. Intraperitoneal glucose (2.0gkg⁻¹) and insulin (1Ukg⁻¹) tolerance tests were performed in overnight-fasted and 4 hour-fasted mice, respectively. For en face analysis, mice were euthanized and perfused with 0.5mM EDTA/PBS. Aortas were dissected, pinned, and stained with Sudan IV. Hearts were paraffin-embedded, sectioned and stained for Oil Red O, CD68, SMA. Images were captured with a CCD camera. Atherosclerosis in the aortic roots and the descending aortas (en face) were quantified by computer-assisted image analysis as described (Shih et al., 1998 (link); Tangirala et al., 1995 (link); Tangirala et al., 1999 (link)). Lesion development is expressed as the percentage of total aortic surface covered by lesions (Bradley et al., 2007 (link)). Cholesterol and triglyceride levels were determined using a commercially available enzymatic kit (Sigma-Aldrich). Blood was collected from the abdominal vena cava. Aliquots of plasma were snap frozen and stored at -80C. (Joseph et al., 2002 (link))