Bead beater 16

Cells were grown to the late exponential phase (OD 600 = 0.7) in BHI at 37 °C in a 5 % CO 2 aerobic atmosphere. The cells were treated with the MIC concentrations of the plant extracts (see Results section) and incubated at 37 °C in a 5 % CO 2 atmosphere for 5 min. The cells were harvested and resuspended with 1 mL of RNAprotect Bacteria Reagent (Qiagen) and incubated for 10 min at room temperature. They were then resuspended in 10 mM Tris-EDTA buffer (10 mM Tris, and 1 mM EDTA; pH 7.5) and subjected to mechanical lysis in a Bead Beater-16 (BioSpec Products, Inc.). The total RNA was isolated using the RNeasy Mini Kit (Qiagen), and the RNA concentration was determined with NanoDrop 2000 (Thermo Fisher Scientific). Next, cDNA was synthesized from 1 µg of the total RNA using SuperScript IV First-Strand Synthesis System (Invitrogen) according to the manufacturerʼs instructions. The target specific primers used for the qPCR were designed with Pri-merQuest Tool software (Integrated DNA Technologies). Their sequences are listed in ▶ Table 3. qRT-PCR was performed using 2 × qPCR MasterMix (with EvaGreen, high ROX) (Coregen) and the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) as follows: one cycle of 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s, and 60 °C for 30 s. All the expression data were normalized to 16S rRNA copy number in each sample.

Free SCFAs were extracted from fecal samples as previously described (27) with slight modifications. Briefly, 0.1 g fecal samples were homogenized in 1 mL diethyl ether containing 5 mmol/L heptanoic acid (used as the internal standard) with a Beadbeater-16 (Biospec, Bartlesville, OK, USA). The tubes were centrifuged at 5000 g for 10 min at 4 °C and the supernatant extracted and used as the SCFA sample. One microliter of supernatant was injected for gas chromatography measurement.
The SCFAs (acetic acid, propionic acid and butyric acid) were quantified by gas chromatography using a gas chromatography system 7890 A equipped with a flame ionization detector (Agilent, Santa Clara, CA, USA). The chromatographic column was an HP-FFAP (25 m, 0.32 mm, 0.5 mm; Agilent). The oven temperature was programmed at 50 °C for 3 min, then increased at 5 °C/min to 140 °C for 1 min, and then increased at 30 °C/min to 240 °C for 3 min. The injector temperature was 230 °C. The injection type was splitless. Nitrogen was used as a carrier gas. Concentrations of SCFAs were determined based on standard curves of an internal standard in terms of micromoles per gram of feces after correction for dilution.

After incubation, PMNL were pelleted by centrifugation and the supernatant collected for further analysis. Total RNA was extracted using Qiazol reagent (Qiagen, Hilden Germany). The cell pellet was placed in 1 mL of Qiazol and tissue was homogenized with a Bead Beater 16 (Biospec, Bartlesville, OK), using two 30-s cycles of the homogenizer at full speed, and placed on ice for 1 min after homogenization. Homogenized samples were centrifuged to remove any remaining cell debris. Chloroform was then added to the homogenized sample, centrifuged at 16,000 × g for 15 min at 4°C, and the aqueous phase removed carefully. Precipitation of RNA was achieved with the addition of ethanol (Decon Labs Inc., King of Prussia, PA), and the subsequent RNA pellet was washed and cleaned using miRNeasy mini spin columns (Qiagen). Genomic DNA was removed during purification with on-column DNase digestion (Qiagen). The RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). All samples had an RNA integrity value >8.0.