Hplc autosampler

In this study, we found that PF at 100 μM exerted a significant neuroprotective effect; thus, we selected this concentration to explore its cell permeability. Briefly, the cells were treated with 100 μM PF for 4, 8 and 24 h to examine the cellular permeability of PF. Following treatment, the cells were washed with Tris-buffered saline and collected in a microcentrifuge tube with ice-cold lysis phosphate-buffered saline (PBS) buffer. The collections were vortexed after being sonicated. Intracellular PF was extracted by centrifugation at 12,000 × g for 15 min at 4°C. The supernatant was then collected and filtered through a 0.22 μm PTFE membrane (Millipore, Milford, MA, USA) prior to injecting into the HPLC autosampler (Shimadzu Co., Kyoto, Japan). The samples were injected into the HPLC system (Shimadzu Co.) equipped with an diamonsil C18 reversed-phase column (I.D., 4.6 mm x 250 mm, 5 μm) at a flow rate of 1.0 ml/min using 30% water (containing 0.1% methanoic acid) and 70% acetonitrile as the mobile phase with a detection wavelength of UV 230 nm.