C57b6 male mice

Nine adult, C57B6 male mice (Jackson Laboratory) were placed on a high fat diet (60% kcal from fat). Four received vertical sleeve gastrectomy surgeries (VSG mice), while the remaining five received no surgery (Control mice). All mice were housed in single cages on a 12-hr light dark cycle, and maintained on ad libitum water.

Eight- to 12-week-old C57B6 male mice (Jackson Laboratory, Strain No. 000664) were used for all experiments. After experimental procedures, mice were euthanized with CO2. All murine experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Emory University and were performed according to the Emory guidelines for the ethical treatment of animals.

Male C57B6 mice (4–5wks; Jackson Labs, Bar Harbor, ME) were injected once with rhPTH 1–34 (50 μg/kg) (Bachem, Torrance, CA) or vehicle (saline) 2h prior to sacrifice. Spleens and long bones were frozen in liquid nitrogen then placed in ice cold methanol containing dueterated internal standards (d₈-5S-hydroxyeicosatetraenoic acid (HETE), d₄-leukotriene (LT) B₄, d₄-prostaglandin (PG)E₂ and d₅-lipoxin (LX) A₄; 500pg each) and homogenized using a PTFE dounce (Kimble Chase). Proteins were precipitated (4°C), solid-phase extracted using Biotage RapidTrace_®⁺(5 (link)), and analyzed using liquid chromatography-ultraviolet-tandem mass spectrometry, QTrap 5500 (ABSciex, Framingham, MA) equipped with an Agilent HP1100 binary pump (Santa Clara, CA). An Agilent Eclipse Plus C18 column (100mm × 4.6 mm × 1.8 μm) maintained at 50°C was used with a gradient of methanol/water/acetic acid of 55:45:0.01 (v/v/v) to 100:0:0.01 at 0.4 ml/min flow rate. Multiple reaction monitoring (MRM) with signature ion fragments for each molecule was used with six diagnostic ions employed for identification, and quantification achieved using calibration curves (5 (link)). Principal component analysis (PCA) was performed using SIMCA 13.0.3 software (Umetrics, Umea, Sweden) following mean centering and unit variance scaling of lipid mediators (LM).