Nefa c test

Serum was collected from blood obtained by cardiocentesis under anesthesia and stored at −80 °C until measurement. Leptin (Leptin ELISA, Millipore, St. Charles, MO, USA), insulin (Mouse Insulin ELISA kit, Shibayagi, Gunma, Japan), glucose (Glucose CII-test, Wako, Osaka, Japan), and non-esterified fatty acid (NEFA) (NEFA C-test, Wako) levels in the sera were measured according to the manufacturers’ protocols.

Plasma non-esterified fatty acid (NEFA), triglyceride, and total cholesterol levels were measured using enzymatic commercial assay kits (NEFA C-Test, Triglyceride E-Test, and Cholesterol E-Test, respectively; Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin, adiponectin, and leptin levels were measured by enzyme-linked immunosorbent assays (ELISA; Mouse Insulin ELISA Kit, Mouse/Rat High Molecular Weight Adiponectin ELISA Kit, and Mouse Leptin ELISA Kit; Shibayagi, Gunma, Japan).

Untreated skin samples were frozen in liquid nitrogen and stored at −80°C until lipid extraction. Adipose tissue was removed from the sections and the remaining skin cut into 0.1-mg pieces. These pieces were homogenized in 50 mM aqueous sodium chloride solution using a Bio Mixer (Nissei Corporation, Tokyo, Japan). Next, a chloroform:methanol (2:1) mixture was added to the lysate in pre-weighed centrifuge tubes, and the suspension centrifuged (2,000 ×g, 4°C, 10 min). The bottom organic layer was removed and allowed to dry overnight. The total lipid weight was calculated by weighing the tubes and subtracting the empty tube weight. Total lipid extracts were dissolved in octylphenol ethoxylate (Wako Pure Chemical Industries, Ltd.) adjusted to 20% (v/v) with 2-propanol (Wako Pure Chemical Industries, Ltd.), and the concentrations of cutaneous cholesterol and fatty acids determined using commercial kits (Cholesterol E-Test, Wako Pure Chemical Industries, Ltd. And NEFA C-Test, Wako Pure Chemical Industries, Ltd., respectively).

The blood glucose was measured with a Glutest sensor (Sanwa Kagaku, Aichi, Japan). The serum levels of triglycerides (Triglyceride E-test, Wako Chemical, Osaka), non-esterified fatty acids (NEFA C-test, Wako Chemical, Osaka), and ketone bodies (EnzyChrom Ketone Body Assay Kit, Bio Assay Systems, CA, USA) were measured according to the manufacturers’ protocols.

Mice were kept in individual cages in a temperature-controlled room at 23 ± 1°C and maintained under a constant 12 h light/dark cycle. Male C57BL/6 mice were purchased from CLEA Japan (Tokyo, Japan). 7-week-old mice were maintained for 7 days on a normal diet (ND) (Research Diet Inc., New Brunswick, NJ, USA) and then divided into two groups of similar average body weight. Each group was maintained on a high-fat diet (HFD) containing 60% kcal fat (Research Diet Inc.) or HFD containing 1% tomato extract (section 2.3) for 10 weeks. This animal experiment was performed under free-feeding conditions. At the end of the treatment period, anesthetized mice were sacrificed by cervical dislocation, and blood and organ samples were collected. Plasma triglyceride (TG), glucose, and non-esterified fatty acid (NEFA) levels were measured using the TG E-test Wako kit (Wako), Glucose CII-test (Wako), and NEFA C-test (Wako), respectively. All animal experiments were approved by the Kyoto University Animal Care Committee (approval code: 28–76).

In order to assess the cell functions and viability, 200 μL of media were withdrawn from the mixing chamber at fixed intervals (0, 4, and 24 h). Biochemical assays were performed to measure the change over time in the metabolite and pro-inflammatory marker levels in the media.
Free Fatty Acids (FFAs) and triglycerides were measured by a colorimetric enzyme assays (respectively NEFA C test-Wako Chemicals GmbH, Germany and Hagen Diagnostica SRL, S. Giovanni V.no (AR), ITALY). Glycerol was determined by a modified Lloyd assay using an automated spectrophotometer Cobas Fara II (Roche) [27 (link)]. IL-6, (eBioscience Dx Diagnostic, Vienna, Austria), E-Selectin, (Boster Biological Technology, LDT, Tema Ricerca, Bologna, Italy), MCP-1 (Life Technologies Italia, Monza-MI, Italy, albumin (Bethyl Laboratories, Montgomery, TX, USA), were determined using ELISA. Other assays and their results are reported in the Supporting Information, S1 File.
At the end of the experiments the cells were fixed and stained with DAPI and phallodin or anti-vWF (all from Thermo Fischer, Italy) and observed with a confocal (Nikon A1, IT) or fluorescent microscope (Olympus X81, IT). Adipose tissue was stained with oil-red stain. The intensity of vWF staining was quantified by image processing as described in the Supporting Information, S1 File.