Labassay nefa kit

Blood samples were centrifuged at 2,000 g for 10 min to obtain serum. Triglyceride (TG), Total cholesterol (TC), high density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were analyzed using Zhuoyue-450 automatic biochemical analysis device (Shanghai Kehua Biotechnology, China). Nonesterified fatty acids (NEFA) levels were determined using the LabAssay™ NEFA kit (WAKO, Japan).

Blood was collected from the retro-orbital venous plexus of overnight-fasted rats. Plasma glucose, free fatty acid, triglyeride and total cholesterol levels were measured using a Wako LabAssay glucose kit (298-65701), LabAssay NEFA kit (294-63601), LabAssay Triglyceride (290-63701) and LabAssay Cholesterol kit (294-65801) (Wako Chemicals USA, Inc.), respectively.

Serum was prepared by leaving the blood to clot undisturbed at room temperature for 30 min and then removing the clot by centrifuging at 2000 g for 10 min at 4 °C. Serum glucose was determined by Glucose Assay Kit (Abcam, Cambridge, UK). Serum high-density lipoprotein (HDL) cholesterol was assayed by Cholesterol, HDL Test, Precipitating Reagent (Stanbio Laboratory, Boerne, TX, USA). Cholesterol was determined with Cholesterol LiquiColor (Stanbio Laboratory, Boerne, TX, USA). Serum insulin was measured by Mercodia Mouse Insulin ELISA kit (Mercodia, Uppsala, Sweden). The HOMA-IR (homeostasis model assessment of insulin resistance) index was calculated as (fasting serum glucose (mmol/L) × fasting serum insulin (mIU/L)/22.5) to assess insulin resistance [19 (link),20 (link)]. Serum and liver triglyceride (TG) were assayed using Triglycerides LiquiColor (Stanbio Laboratory, Boerne, TX, USA). Non-esterified fatty acid (NEFA) was measured by LabAssay NEFA kit (Wako Pure Chemical Industries, Osaka, Japan).

Mammary glands from 8- to 12-wk-old virgin female mice or mice at lactation day 2 were isolated. The minced tissue was digested in RPMI 1640 with 25 mM HEPES, 5% FBS, 1% PSQ, 300U/mL Collagenase III (Worthington) for 2 hours at 37°C. After lysis of the red blood cells, single cell suspension was obtained by sequential incubation with 0.25% Trypsin-EDTA for 5 min and 0.1 mg/mL DNase I (Sigma) for 5 min at 37°C with gentle pipetting, followed by filtration through 40-μm cell strainers. The antibodies used for flow cytometry were: PE-conjugated CD31, CD45, TER119 (BD PharMingen), CD24-PE/cy7 and CD29-APC (Biolegend). All sorting was performed using FACSAria or FCASJazz (Becton Dickinson). The purity of sorted population was routinely checked and ensured to be >95%. Cells were harvested for quantitative RT-PCR experiments. For insulin treatment experiment, primary mammary epithelial cells were seeded in DMEM (Invitrogen) supplemented with 10% FBS. Adhered cells were treated with 10μg/mL insulin for 24 hours and were harvested for quantitative RT-PCR experiments. Cytosolic triacylglycerol and nonesterified fatty acid in primary mammary epithelial cells were quantified following manufacturer's protocols (Triglycerides kit, Shanghai KEHUA bio-engineering CO., and LabAssay NEFA kit, Wako).

Glu, TG, and TCH levels were measured using standard methods with a Dri-Chem 7000 V (FUJI FILM). Insulin levels were determined using an insulin assay kit (Morinaga Institute of Biological Science Inc., Tokyo, Japan). Serum free fatty acid (FFA) levels were measured using a LabAssay NEFA kit (Wako Pure Chemical Industries Inc., Osaka, Japan).