Ptfe dounce

Supernatant from the inflammatory lesions were placed in ice cold methanol containing deuterated 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 allowed to precipitate (4°C), and lipid mediators were extracted using C18 solid-phase cartridges and a Biotage RapidTrace®. Measurement of lipid mediators was carried out by liquid chromatography-tandem mass spectrometry using a QTrap 5500 (ABSciex, Framingham, MA) equipped with a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector (Shimadzu, Kyoto, Japan). 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 reactions monitoring (MRM) was used to monitor lipid mediator profiles with more than 60 bioactive products from specific biosynthetic pathway including their pathway markers. Identification was carried out with signature ion fragments for each target lipid mediator (pro-inflammatory mediators PG, LT as well as SPM) using a minimum of six diagnostic ions. Quantification was achieved using calibration curves (Colas et al., 2014 (link)).

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).