Agilent 6230 tof ms system

Extractions were performed according to a previous report by Hatakeyama et al. [35 (link)]. Briefly, 1.0 × 10⁶ astrocytes or MDA231 were collected in one tube. Cells were detached by trypsin-EDTA treatment and washed twice with 5% mannitol. Cell pellets were suspended in 1 mL of methanol, sonicated for 30 s, and vortexed with 1 mL of chloroform and 0.4 mL of ultra-pure water for 30 s. After centrifugation at 2300× g at 4 °C for 5 min, the aqueous layers were filtered through a Nanosep/3K (3-kDa cut-off) filter (Nihon Pall Ltd., Tokyo, Japan) at 9100× g at 4 °C for 2.5 h to remove proteins and phospholipids. The resultant filtrates were used as ionic metabolites; they were lyophilized and dissolved in 25 μL ultra-pure water prior to CE-TOFMS analysis. CE-TOFMS was carried out using an Agilent 7100 CE system equipped with an Agilent 6230 TOF-MS system (Agilent Technologies, Santa Clara, CA, USA). Raw data were processed using the Mass Hunter software (Qualitative and Quantitative Analysis, Agilent Technologies, Santa Clara, CA, USA) for metabolite quantification.

Metabolome analysis was performed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). Cellular extracts were prepared as described previously [22 (link)]. Briefly, HK-2 cells were plated at a density of 1.0 × 10⁶ cells in a 100-mm dish and incubated for 2 days prior to treatment with sodium-β-hydroxybutyrate. Cells were washed twice with 5% mannitol and detached by treatment with trypsin–EDTA. Cell pellets were resuspended in 1 ml methanol and sonicated for 30 s. Cell suspensions were mixed with 1 ml chloroform and 0.4 ml ultra-pure water and vortexed for 30 s. After centrifugation at 2300×g at 4 °C for 5 min, the aqueous layers were filtered through an UltrafreeMC-PLHCC (5-kDa cutoff) filter (Human Metabolome Technologies, Yamagata, Japan) at 9100×g at 4 °C for 2.5 h to remove proteins and phospholipids. The filtrates were lyophilized and dissolved in 25 μl ultra-pure water. CE-TOF-MS analysis was carried out using an Agilent 7100 CE System equipped with an Agilent 6230 TOF-MS System (Agilent Technologies, CA). Raw data were processed with MassHunter software (Qualitative and Quantitative Analysis, Agilent) for the quantification of metabolites.