Vacufuge plus

Three types of extracts were obtained from AmH, AmL and AmIV plant material by mixing 250 mg of powdered plant with 5 mL of either 96% ethanol (96% EtOH extracts), 50% ethanol (50% EtOH extracts) or water (H₂O extracts) and extracted using the digital ultrasonic water bath CD-482 (Hotair, Zawiercie, Poland) operating for 30 min at room temperature. The extracts (in triplicate) were evaporated to dryness using a Concentrator plus/Vacufuge^® plus (Eppendorf, Hamburg, Germany) at the temperature of 30 °C for 96% EtOH extracts and at 45 °C for 50% EtOH and H₂O extracts. Dried residues were stored at −80 °C upon analysis.

The protein spots were carefully excised from gels and then de-stained for 30 min in wash buffer (100 μL, 25 mM NH₄HCO₃/50% acetonitrile (v/v)). Washed-out gel-spots (dehydrated in 100% acetonitrile) were dried completely with a centrifuge (Vacufuge plus, Eppendorf, Hamburg, Germany) and then incubated in 15 ng/μL trypsin and 25 mM NH₄HCO₃ (37 °C, 16 h). The peptides were incubated in trifluoroacetic acid (20 μL, 0.1% (v/v), 37 °C, 40 min) after digestion. The above extraction procedures were repeated by using 50% acetonitrile/0.1% trifluoroacetic acid (v/v). Sediments were washed in trifluoroacetic acid and then vacuum freeze-dried for further analysis. The proteins were identified by using AUTOFLEX II TOF-TOF mass spectrometer (autoflex™ speed, Bruker Daltonik, Bremen, Germany). Samples in 1 μL of buffer (50% acetonitrile and 5 mg/mL α-CHCA in 0.1% trifluoroacetic acid) were loaded on plate (AnchorChip, 384-MPT).
Each crystallized sample was washed by using 0.1% trifluoroacetic acid for removing salt ions. Protein identification was performed by peptide mass fingerprinting (PMF), searching the Mascot (2.2 version, Matrix Science, London, UK), and matched with a sheep (Ovis aries) family in the Uniprot database (https://www.uniprot.org/ (accessed on 8 October 2021)), correspondingly.

For cell counting, 200 mg aliquots of the samples were processed and stained as described by Vandeputte et al. (2017 (link)) followed by flow cytometric analysis using a BD FACSCanto II with FACS Diva V8.0.1 software (BD Biosciences). A side scatter of 2,000 was set as acquisition threshold. All other instrument and gating settings were in accordance with the method described by Vandeputte et al. (2017 (link)) and were kept constant for all samples. To obtain bacterial concentrations, the total number of events in the cell gate was divided by the sample volume, which was determined by weighing each tube before and after acquisition.
Stool moisture content was determined in duplicate on 200 mg homogenized fecal material as the percentage of mass loss upon vacuum concentration for 5 h at 60°C in a Vacufuge plus (Eppendorf) using the “AQ” setting.

Free phenolic molecules from the barley whole meal flour were extracted from adapted protocols of previously published studies using an ethanol 90% solution with modifications [27 (link),34 (link)]. One gram of powder was extracted with 15 mL of ethanol 90% and sonicated in a water bath at 70 °C for 1 h with periodical inverting of tubes. The supernatant was filtered, using Whatman#4 filter paper, into new tubes and concentrated under a vacuum at 60 °C for 2 h using Eppendorf Vacufuge Plus (Eppendorf, Germany). The residue was suspended in 500 μL HPLC-grade methanol, then sonicated for 5 min at 50 °C until complete solubility. This methanolic extract was centrifuged at 12,000× g rpm for 20 min, and 140 μL aliquot was transferred into vial inserts prior to HPLC analysis.

Tamoxifen (Sigma T5648-5G, final concentration 2 mg/mL) was dissolved in ethanol (40 mg/mL) and mixed with corn oil (375uL Tamoxifen in EtOH/750uL corn oil) in 1.5 mL microcentrifuge tubes. After thorough vortexing, ethanol was evaporated by spinning in Eppendorf Vacufuge Plus on V-AL (vacuum-alcohol) setting at 3 °C for 20 min (or until all EtOH had evaporated). Mice received 5 daily injections of Tamoxifen (200 mg/kg, IP) beginning at 60–90 days of age and were given 10–14 days to recover before performing additional procedures or testing. All animals (except where specified) were treated with Tamoxifen, such that floxed mice without CamkIIα-CreER^T2 were used as controls. During Tamoxifen treatment, mice were housed in ventilated cages (5 × 7 × 14 in.) in a closed-circuit atmosphere rack (Techniplast Easy Flow #BOX110EFUL) and transferred to standard housing thereafter.