Type I interferon protects neurons from prions in in vivo models

Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Mori, Tsuyoshi; Atarashi, Ryuichiro; Nishida, Noriyuki

RO8191-treated and -untreated mice brains and spleens at 100 dpi and terminal phase were homogenated with PBS. Then 20% homogenate samples were mixed with 99% methanol in a 4-fold volume and were incubated at À80 C for 1 h. The standard samples were prepared from serial dilutions (0.1, 1, 10, 100 and 1000 nM) of compounds mixed with 20% normal brain homogenate. After centrifugation at 20 000g for 15 min, supernatants from the sample tissues and standards were collected, and then subjected to LC-MS/MS. LC-MS/MS was performed by an acquity UPLC-I class system coupled with a Xevo TM TQ-S triple quadrupole-mass spectrometer (Waters). Under UPLC conditions, 5 ml of sample volume was delivered to the ACQUITY UPLC BEH C18 column (1.7 mm, 1.0 Â 100 mm, Waters) for reversed-phase chromatography, with the mobile phase consisting of solvent A (5 mM ammonium formate in methanol) and solvent B (ultra-pure water) at a flow rate of 0.35 ml/min at 40 C. In the gradient elution, solvent A was used in the order corresponding to ratios of 10%, 10%, 95% and 10% at each time point (0, 1.5, 2.0 and 4.5 min, respectively). To detect the separated compound, the tandem mass spectrometer was set in ESI positive ionization mode and in multiple reaction-monitoring (MRM) mode. Collision energy and cone voltage were 3000 V and 50 V, respectively. Cone and desolvation gas flow rates, and the desolvation temperature were set to 150 and 650 l/h, and 350 C, respectively. The system was tuned to monitoring the scan type of MRM with three channels set to 373.98 to 265.00, 373.98 to 331.98 and 373.98 to 347.06 m/z transition (monitor range, precursor ion to product ion). From the MRM data, the concentration of RO8191 was acquired by analysis through MassLynx software (Waters).