Biophotometer

Total RNA was extracted from heart samples with the use of the acid guanidiniumthiocyanate-phenol-chloroform method thanks to a Rneasy Midi kit (QiagenS.p.a, Milano, Italy) as described by the manufacturer. RNA concentration and purity were evaluated spectrophotometrically (BioPhotometer Eppendorf, Milan, Italy) and by electrophoresis of samples on Gel Star Stain (Lonza Rockland Inc., ME, USA) agarose gels. Only samples with spectrophotometric 260/280 nm ratios of 1.8–2.1 and clear 28S and 18S ribosomal RNA bands resulting from electrophoresis were used. A known amount of total RNA (Ambion, Inc., Austin, TX, USA) was used as marker. The RNA samples were stored at −80°C for use in gene expression studies.
Following DNAse treatment (RNase-Free DNase Set, QiagenS.p.A), first-stand cDNA was synthesized by IScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) starting from about 1 μg total RNA as template. Reverse transcriptase reaction sequence consisted of an incubation step at 25°C for 5 min, followed by three different cycles at 42°C for 30 min and 45–48°C for 10 min, in order to better separate the strands. The reverse transcriptase enzyme was inactivated by heating to 85°C for 5 min. The cDNA samples obtained were placed on ice and stored at 4°C for a maximum of 1 month.

Total RNA isolation from tissue homogenates of rat brains (frontal cortex, hippocampus) was carried out using TriPure Isolation Reagent (Roche), according to the manufacturer's protocol. The integrity of RNA was visually assessed electrophoretically and spectrophotometrically (BioPhotometer Eppendorf); 1 μg of total RNA from all samples was used for reverse transcription into cDNA using Transcriptor First Strand Synthesis Kit (Roche), according to the manufacturer's protocol. Then, the samples were stored at −20°C or used directly for quantitative real-time PCR (qRT-PCR).

Total RNA isolation was carried out using TriPure Isolation Reagent (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s protocol. The integrity of RNA was visually assessed by conventional agarose gel electrophoresis and the concentration was evaluated by measuring the absorbance at 260 and 280 nm in a spectrophotometer (BioPhotometer Eppendorf, Hamburg, Germany). RNA samples were stored at −80 °C until use. 1 µg of total RNA from all samples was used for the reverse-transcription into cDNA using the Transcriptor First Strand Synthesis Kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s protocol. Obtained cDNA samples were stored at −20 °C or used directly for the quantitative real-time PCR (qRT-PCR) reaction.

Total RNA isolation from the rats brain tissues homogenates (frontal cortex, hippocampus) was carried out using TriPure Isolation Reagent (Roche) according to manufacturer's protocol. The integrity of RNA was visually assessed by a conventional agarose gel electrophoresis and the concentration will be evaluated by measuring the absorbance at 260 and 280 nm in a spectrophotometer (BioPhotometer Eppendorf). RNA samples were stored at −80°C until use. The 1 μg of total RNA from all samples was used for the reverse transcription into cDNA using Transcriptor First Strand Synthesis Kit (Roche) according to manufacturer's protocol. Obtained cDNA samples was stored at −20°C or used directly for the quantitative real-time PCR (qRT-PCR).

Cell growth was measured using the optical density at 600 nm (OD₆₀₀) with a BioPhotometer plus a UV-Visible spectrophotometer (Eppendorf, Hamburg, Germany). Biomass concentration was determined by the correlation of dry cell weight (DCW) with OD₆₀₀ as described in a previous report^{18 (link)}. The concentration of formate was measured using high-performance liquid chromatography (HPLC) equipped with a UV detector and an RSpak KC-811 column (Shodex, Tokyo, Japan) at a flow rate of 1.0 ml min⁻¹ with a mobile phase of 0.1% (vol/vol) H₃PO₄. The concentration of H₂ in the headspace was measured by sampling the headspace gas (100 μl) using gas-tight syringes using an YL6100GC gas chromatograph (GC) (YL Instrument Co., Anyang, Republic of Korea) equipped with a Molsieve 5A column (Supelco, Bellefonte, PA, USA), a Porapak N column (Supelco), a thermal conductivity detector and a flame ionization detector. Then, the production rate of hydrogen (mmol per liter of medium) was calculated. Argon was used as a carrier gas at a flow rate of 30 ml min⁻¹. The total volume of outlet gas in a bioreactor was measured using a wet gas meter (Shinagawa, Tokyo, Japan)

Cells of Bifidobacterium bifidum PRL2010 were recovered from an overnight culture, and turbidity was measured at 600nm using a biophotometer (Eppendorf). A growth tube containing 6ml of MRS was inoculated with active viable bacterial cells diluted to an OD_600nm of ~1.0, obtaining a final inoculum with an OD_600nm of ~0.1. PMAxx^™ (Biotium Inc., CA, United States) was added at a concentration of 100μM. Cultures were grown in biologically independent triplicates, and the resulting growth datasets were expressed as the means from these replicates. Moreover, positive growth controls without PMAxx^™ were performed. Cultures were incubated under anaerobic conditions at 37°C for 24h. Cell growth was monitored using a Thoma cell counting chamber according to the producer’s instructions (Herka).

Frozen tissue samples (−80 °C) from the intestinal cecum were used for the study of microbiota. Genomic DNA (gDNA) was extracted from 200 mg of frozen cecum feces using E.Z.N.A.^® DNA Stool Kit (VWR, Madrid, Spain) and provided at least 200 μL of genomic DNA. These gDNA samples were then quantified using a BioPhotometer^® (Eppendorf, Madrid, Spain) and their concentrations were finally diluted to 6 ng/μL. The diluted samples were used for performing polymerase chain reaction (PCR) amplification, following the protocol of the Ion 16 Metagenomics kit (Thermo Fischer Scientific, Madrid, Spain) [36 (link)].
PCR amplification products were used to create a library using the Ion Plus Fragment Library kit for AB Library Builder System (Thermo Fischer Scientific, Madrid, Spain), with sample indexing using the Ion Xpress Barcode Adapters 1–96 kit (Thermo Fischer Scientific, Madrid, Spain).
Template preparation was performed using the ION OneTouch 2 System and the ION PGM Hi-Q OT2 kit (Thermo Fischer Scientific, Madrid, Spain). Metagenomics sequencing was performed using the ION PGM Hi-Q Sequencing kit (Thermo Fischer Scientific, Madrid, Spain) on the ION PGM System. The chips used were the ION 314 v2, 316 v2 or 318 v2 Chips (Thermo Fischer Scientific, Madrid, Spain) with various barcoded samples per chip.