Precellys 24 tissue

All solutions were prepared with ultrapure water containing 0.5% metaphosphoric acid. Ascorbic acid, Ascorbic acid-¹³C₆ and metaphosphoric acid were obtained from Sigma Aldrich (St-Louis, MO). Formic acid, methanol and acetonitrile of LC-MS grade were purchased from Fisher Scientific (Pittsburgh, PA). Liver tissues (approximately 100 mg) were homogenized with the soft tissue CK14 lysis kit from VWR (Radnor, PA) in a Precellys 24 tissue homogenizer (Bertin Instruments, France) with 1 mL of ice-cold metaphosphoric 0.5% solution. Samples were then centrifuged at 21,380 g for 10 min at 4 °C. The supernatants were then passed through a Sep-Pak cartridge CN and a 0.2 μm GHP Acrodisc filter (Waters, Milford, MA). Filtered samples were diluted appropriately and Ascorbic acid-¹³C₆ solution was added to all samples as an internal standard before analysis.

Murine colonic tissues, or ENS cultures, were lysed in RA1 buffer with the “Precellys 24” tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). The lysate was transferred to a Nucleospin RNA II filters column (Macherey Nagel, Hoerdt, France). The proteins were recovered from the flow-through and stored at −20 °C. The RNAs were eluted from the columns and treated with DNase according to the manufacturer’s recommendations and stored at −20 °C.

Dura mater was isolated as mentioned above without any transcardial perfusion. Dura mater was removed from the attaching calvarial bone by submerging the tissue in ice-cold PBS and peeling the organ with fine forceps. Tissue was mechanically dissociated using a Precellys 24 tissue homogenizer (Bertin Technologies) until cells were completely lysed. RNA was extracted using RNeasy Micro kit (Qiagen) or Trizol RNA extraction kit (Invitrogen). A total of 1 µg of extracted RNA was transcribed into cDNA using GoScript Reverse Transcription Kit (Promega). Quantitative real-time PCR was performed using FastStart SYBR Green Master mix (Roche) and S1000 Thermocycler (Bio-Rad) with the indicated primers. The primers were designed using Primer-BLAST or adopted from previously published studies: Vegfa (5′-TGCCAAGTGGTCCCAGGCTGC-3′; 5′-CCTGCACAGCGCATCAGCGG-3′); Vegfc (5′-GAGGTCAAGGCTTTTGAAGGC-3′; 5′-CTGTCCTGGTATTGAGGGTGG-3′); Pgf (5′-TCTGCTGGGAACAACTCAACA-3′; 5′-GTGAGACACCTCATCAGGGTAT-3′). Gapdh (5′-AGGTCGGTGTGAACGGATTTG-3′; 5′-TGTAGACCATGTAGTTGAGGTCA-3′) was used as a reference gene and the results were presented as relative expressions to control. Primer reaction specificity was confirmed by melting curve analysis. Relative gene expression was analyzed by ΔΔCt method using the CFX Manager software (Bio-Rad).

RNA extraction was performed as described previously. Tissue was dissociated using Precellys® 24 tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and total RNA purified using the automated QIAcube with RNeasy Fibrosis Mini kit (Qiagen, CA, United States) according to the manufacturer’s instructions. The RNA integrity was assessed with the Bioanalyzer 2,100 system (Agilent, CA, United States), and RIN values > 8 accepted as suitable for PCR profiling. RNA concentrations were then measured using the Nanodrop 2000 (Labtech International, East Sussex, United Kingdom).