Dionex ics 5000 plus

We cut a fresh leaf into two small pieces across the vein and dried them in an oven at 65 °C, resulting in about 30 mg in dry weight. The sample was homogenized into fine powder with 1 mm glass beads at 30 revolutions/second for 2 min in TissueLyser II (QIAGEN). We then weighed out 5 to 6 mg of the powder sample, and mixed it well in 1.5 mL Milli-Q water. The mixture was then incubated for 1 h, sonicated for 20 min, filtered with a 0.2 μm filter tip, and submitted for IC analysis (Dionex ICS-5000 plus, Thermo Fisher Scientific) to quantify total phosphates. An AG11HC guard column was used along with chromatographic separation using a Dionex CS12A, Ion Pac (2 × 250 mm) analytical column at 20.5 °C, and an injection volume of 25 μL. The elution of anions was achieved with a concentration gradient of 6 mM to 21.5 mM in 16.5 min, 21.5 to 60 mM in 6.5 min, and at 60 mM for 3 min, then re-equilibrated at 6 mM for 8 min at a flow rate of 0.33 ml/min. Standard anions (Dionex, Thermo Fisher Scientific) were used, with ion quantification using commercial software (Chromeleon 7.2 SR4, Thermo Fisher Scientific).

Daily gut model samples from all vessels of each model were assessed by ion chromatography for trehalose and glucose concentrations. Samples were tested in a blind fashion. Gut model samples were centrifuged at 15,000 rpm for 10 mins, and the supernatant was further centrifuged using millipore centrifugal filter units with a 50 kDa and 3 kDa cut off to remove proteins and bigger macromolecules. Samples were diluted 1:10 in ultrapure water and analysed by ion chromatography. 25 µl of sample was injected on a Dionex ICS-5000 plus ion chromatography system (Thermo fisher Scientific, USA). The separation was carried out on a Dionex CarboPac PA1 Analytical Column (4 x 250 mm) with a Dionex CarboPac PA1 Guard Column (4 x 50 mm). The column temperature was set at 30°C. Elution was performed using three elutants; A: ultrapure water, B: 300 mM sodium hydroxide and C: 300 mM sodium hydroxide with 1500 mM sodium acetate, with a flow rate of one ml/min. The linear gradient condition is shown in Supplementary Table 5. The analytes were detected by using an electrochemical detector.