5 kda cut off filters

Cells were collected for metabolome analysis at the indicated infection times. Approximately 2.5 × 10⁶ cells were infected with each V. parahaemolyticus strain at each given time point at an MOI of 50:1. The supernatant was removed, and cells were washed twice with 5 ml of cold 5% mannitol solution. Metabolic activity was rapidly quenched by adding 0.5 ml methanol containing internal standards (100 μM methionine sulfone and camphor 10-sulfonic acid). Intracellular metabolites were extracted using a solvent extraction method by mixing homogenates with 400 μl chloroform and 200 μl Milli-Q water. The mixture was centrifuged (5,000 rpm, 4°C, 5 min). Subsequently, the aqueous layer was filtered using 5-kDa-cutoff filters (Millipore, Bedford, MA) and centrifuged (10,000 rpm, 4°C, 6 h). The filtrate was dried using a vacuum evaporator (4,000 rpm, 4°C, 4 h) and reconstituted in 50 μl Milli-Q water containing spiked internal standards (25 mM [each] 3-aminopyrrolidine and trimesic acid) before analysis.
CE-TOF/MS was performed using an Agilent CE capillary electrophoresis system coupled with an Agilent 6210 time of flight mass spectrometer (Agilent Technologies, Palo Alto, CA) by Human Metabolome Technologies, Inc. (HMT; Tsuruoka, Japan), as described previously (52 (link)).

The cecum luminal content was immediately frozen in liquid nitrogen and stored at −80 °C until metabolite extraction. Sample tissues were weighed and completely homogenized in 0.5 mL ice-cold methanol containing 50 μM methionine sulfone and camphor-10-sulfonic acid as internal standards. The homogenates were mixed with 0.5 mL chloroform and 0.2 mL ice-cold Milli-Q water. After centrifugation at 2300× g for 5 min, the supernatant was centrifugally filtrated through 5-kDa cut-off filters (Millipore, Bedford, MA, USA) at 9100× g for 4–5 h to remove proteins. The filtrate was centrifugally concentrated in a vacuum evaporator, dissolved with Milli-Q water, and analyzed by capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE-TOFMS).
CE-TOFMS analysis was performed using an Agilent CE system combined with a TOFMS (Agilent Technologies, Palo Alto, CA, USA) as reported by previously [24 (link),26 (link),27 (link)]. Each metabolite was identified based on a reference which containing internal standards including 110 metabolites (H3304-1002, Human Metabolome Technology (HMT), Inc., Tsuruoka, Japan) to m/z and migration time, and quantified by peak area.

Cecal contents from 13 weeks old mice which stored at −80°C were weighed and completely homogenized in 500 µl methanol containing 50 µM methionine sulfone and camphor-10-sulfonic acid as internal standards. The homogenates were mixed with 500 µl chloroform and 200 µl Milli-Q water. Samples were centrifuged (2,300 g, 5 min, 4°C), and then the supernatant was centrifugally filtered using 5-kDa cut-off filters (Millipore, Bedford, MA) until all was filtered (9,100 g, 4°C). The filtrate was centrifugally concentrated in a vacuum evaporator, dissolved in Milli-Q water and analyzed by capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE-TOFMS).
CE-TOFMS analysis was performed by an Agilent CE system combined with a TOFMS (Agilent Technologies, Palo Alto, CA) as reported by Human Metabolome Technologies Inc. (HMT, Tsuruoka, Japan).^{(11 (link),12 (link))} Each metabolite was identified and quantified based on the peak information including m/z, migration time and peak area.