Tsq 7000

MEFs, WT and nCDase-knockout (1 × 10⁶ cells), were seeded in 10 cm² dishes and treated with 2DG/AA for the indicated times. Cells were harvested, washed two times in 5 ml of ice-cold PBS and protein concentration determined via the BCA assay. 1.5 mg of cellular protein was pelleted and snap-frozen in liquid nitrogen. Quantification of sphingolipid species was performed by the Lipidomics Core Facility at the Medical University of South Carolina (MUSC) on a Thermo Finnigan TSQ 7000, triple-stage quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode as described in [33 (link)]. Data were normalized to total lipid phosphate level.

BMMs (3×10⁶ cells) were lysed in 150 μL RIPA protein lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Periodontal tissues from mice were homogenized in 1 mL RIPA protein lysis buffer. Sphingolipids in cell lysate, tissue lysate, or serum were extracted from the samples by the Lipidomics Core Facility using the Bligh Dyer technique. Sphingolipid analysis was performed using Electrospray Ionization /Tandem Mass Spectrometry (ESI-MS/MS) on a Thermo Finnigan TSQ 7000 triple quadruple mass spectrometer. This technique was previously described by Bielawski et al. (22 (link)). The sphingolipid levels in the cell lysate were normalized to protein concentrations in samples.

RAW264.7 cells were collected, fortified with internal standards, extracted with ethyl acetate/isopropyl alcohol/water (60:30:10, v/v/v), evaporated to dryness, and reconstituted in 100 μl of methanol. Simultaneous ESI/MS/MS analyses of sphingoid bases, sphingoid base 1-phosphates, CERs, and SMs were performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring positive ionization mode. The phosphate contents of the lipid extracts were used to normalize the MS measurements of sphingolipids. The phosphate contents of the lipid extracts were measured with a standard curve analysis and a colorimetric assay of ashed phosphate (24 (link)).

Nuclear magnetic resonance (NMR) spectroscopy was performed with a Varian MercuryPLUS (300 MHz) spectrometer by taking an average of 128 scans (delay 5 s) using appropriate solvents (CDCl₃ or D₂O). Gel permeation chromatography (GPC) was performed on a Waters system with Waters 1525 Binary Pump and Waters 2414 differential refractive index detector utilizing two highly efficient PolySep GFC columns (elution range 3k to 400k Da). An aqueous solution containing 0.1 M NaNO₃ and 0.01% (w/v) NaN₃ was filtered and used as the eluent at a flow rate of 1 mL/min at 25 °C. The molecular weight calibration was performed with monodisperse linear poly(ethylene oxide) (Polymer Standard Service). For molecular weights, the entire signal of a major peak including its shoulder at a lower retention volume was integrated. Mass spectrometry was done on a ThermoFinnigan TSQ 7000 triple-quadrapole instrument that was equipped with an electrospray ionization (ESI) source. Glycomonomer samples (1 mg/mL) in a 1:1 (v/v) methanol/water solution containing sodium chloride (1 mg/mL) were injected into the ESI source at a rate of 10 µL/s. All data were analyzed using Xcalibur (FisherScientific, Inc.) software.