Millex mp

Initially, a small amount of solid compound (generally 0.5–1.5 mg) was measured into a 1.7-ml Eppendorf tube. PBS (pH 7.4) was added to give a maximum final concentration of 1 mg ml⁻¹ of compound. The compound was vortexed for ∼30 s before being placed into a bath sonicator (Cole Parmer, ultrasonic cleaner) for 1 h. Longer incubation times (up to 24 h) were performed with select compounds and no difference in solubility was observed; thus, 1 h was used for all subsequent testing. The tubes were vortexed again for 30 s before being centrifuged at maximum speed (13,000 × g) for 10 min. The supernatant was then filtered through a 0.22-μm syringe filter (Millipore Millex MP). The filtrate was then analysed by liquid chromatography–mass spectrometry (λ=254 nm, electrospray ionization–time-of-flight in positive mode, Agilent Technologies 6230 TOF LC/MS). The filtrate was diluted 1:2 and 1:4 and all three samples (1 × , 0.5 × and 0.25 × ) were analysed in triplicate. Three independent replicates of each compound were performed. A calibration curve for each compound was generated from 1 to 40 μM by dissolving the compound in DMSO and making dilutions of the stock in DMSO. The calibration curve (measured by ultraviolet absorbance) was linear over this range. The concentration of the samples was calculated based on the calibration curves.