Millipore ultrafree mc

The LMW fraction of the PCFs was analysed as described previously for the plasma/serum peptidome [14 (link)]. Briefly, a mix of 100 μl PCF + 300 μl 25% acetonitrile in 25 mM NH₄HCO₃ was centrifuged at 5000 x g, at 15°C, for 90 minutes through a 30 kDa cutoff filtration membrane (Millipore Ultrafree-MC, pore size 30000, Sigma-Aldrich), and then washed twice with 25% acetonitrile prior to use. Next, 100 μl 25% acetonitrile was applied to the filter and again centrifuged for 60 min. Finally, the combined filtrates were centrifuged at 5000 x g, at 15°C, for 120 minutes through the 5 kDa cutoff filtration membrane (Millipore Ultrafree-MC, pore size 5000), and the <5 kDa fraction was lyophilized (Speed Vac) in siliconized microtubes (Clear-view Snap-Cap microtubes, size 1.5 mL, low retention, Sigma-Aldrich) and stored at –80°C for further use. Prior to LC-MS analysis, samples were dissolved in 0.1% trifluoroacetic acid (TFA). MS/MS data was acquired from pooled PCF samples prepared by combining 10 μl of fluids belonging to given cyst type from the study samples.

The water-holding capacity (WHC) was evaluated according to the method described by Kim et al [9 (link)]. Approximately 0.5 g sample was placed on a round plate in a Millipore tube (Millipore Ultrafree-MC; Millipore, Bedford, MA, USA) and heated in a water bath at 80°C for 20 min. It was cooled to 23°C and centrifuged (2,000×g) at 4°C for 10 min to measure the water loss.