Speedvac concentrator system

Cholesterol and cholesteryl esters were quantified by reverse-phase HPLC. Lipids were extracted from macrophages using methanol and hexane [42 (link)]. The upper hexane layer was collected and evaporated at ambient temperature in a SpeedVac Concentrator System (ThermoFisher). The residue was redissolved in the mobile phase (acetonitrile/propan-2-ol (30/70, v/v)) and injected into a C18 column in an HPLC (PerkinElmer 200 series) [42 (link)]. Cholesterol and cholesteryl esters were detected at 210 nm. The identities of the peaks were confirmed by mass spectrometry (results not shown) and quantified using standards of pure chemicals from Sigma.
Cysteamine in mouse plasma samples cells was measured using 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate by reverse-phase HPLC with a C18 column and a fluorescent detector [43 (link)]. Tri-n-butylphosphine was used to release protein-bound thiols and reduce oxidised thiols.

Cells were exposed to normoxia (21% O₂) in a normal CO₂ cell culture incubator or hypoxia (5% or 1% O₂) in a hypoxia chamber (Billups-rothenburg, Del Mar, CA). Sterile water was placed inside the chamber to maintain moist conditions. Hypoxic conditions were created by filling the chamber with 5% or 1% O₂, 5% CO₂ and balanced N₂. The gas was passed through the chamber at 1–2 psi for 3 min, and the chamber was sealed and placed in a 37 °C cell culture incubator. The oxygen concentration in the chamber was monitored using an oxygen sensor (OxyCheq, Marianna, FL). Every 2 days, media were replaced with fresh media that were degassed using a SpeedVac Concentrator system (Thermo Fisher Scientific, Waltham, MA).

Lipids were extracted from oxidised LDL for HPLC analysis using methanol and hexane (Kritharides et al., 1993 (link); Satchell and Leake, 2012 (link)). The upper hexane layer was collected and evaporated at ambient temperature in a SpeedVac Concentrator System (ThermoFisher), the residue was dissolved in the relevant mobile phase before injection into the HPLC. Lipid species were separated by reverse phase HPLC in a Waters C18 column (250 mm × 4.6 mm, 5 μm particle size, 5 μm guard column) with a Agilent 1100 HPLC system. Cholesteryl esters were detected at 210 nm using an acetonitrile/2-propanol/water mobile phase (44/54/2, by volume) and a flow rate of 1.2 mL/min. Cholesteryl linoleate hydroperoxide and 7-ketocholesterol were detected at 234 nm using an acetonitrile/2-propanol/water mobile phase (50/48.8/1.2, by volume) and a flow rate of 1 mL/min. The identities of the peaks were confirmed by mass spectrometry (data not shown), and the lipids were quantified using commercially available standards.