Minilys tissue homogenizer

Composite samples of 5 hempseeds were ground in a Precellys 2 ml Tube, contained in the grinding kit MK28 with 2.8 mm steel beads, using the Minilys Tissue Homogenizer (Bertin Technologies, France). The DNA was extracted with the NucleoSpin Plant II Kit (Macherey-Nagel, Germany). Based on the measured concentration, the extracts were diluted to an operating concentration of 5 ng/µL for subsequent PCR analysis.

Each ventricular tissue was weighed and added to lysis buffer containing (1% Noidet P-40/IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS in 1x PBS) and one tablet of each proteases and phosphatases inhibitor cocktails at a ratio of 10 μL per mg of tissue. The mixture was kept on ice. The biopsies were then homogenized at high speed for 12 sec (twice) using Minilys tissue homogenizer (Bertin technologies, France) which involved the use of bead beating technology. The homogenate was left 30 minutes shaking on ice, at 4°C and then centrifuged at 10,000 g for 10 minutes at 4°C and the supernatant was collected. Protein concentration in the supernatant was determined with the total protein kit, Micro Lowry (Sigma, UK). Bovine serum albumin (BSA) at a stock concentration of 5 mg/mL was used to obtain a standard curve. For each sample, 400 μL of Lowry reagent was added and incubated at room temperature for 30 min, followed by adding 200 μL of Folin-Ciocalteu reagent incubated for 30 min. The absorbance was measured at 750 nm using spectrophotometer (Jenway7305, UK). Protein concentration in samples was determined by interpolating the densities from the standard reference curve.

To test the relative protection conferred by immunization with Typbar TCV^® or Typhim Vi^® admixed with and without Turbo adjuvant, mice were infected with a chimeric strain of S. Typhimurium (strain RC60) that expresses the S. Typhi genes necessary for ViPS synthesis, export, and regulation as in S. Typhi (23 (link)). This protection model was previously validated in several mouse systems (21 (link), 24 (link)–26 (link)). In brief, Strain RC60 was grown to an OD⁶⁰⁰ of ~1.0 in Luria–Bertani broth containing 10 mM NaCl. The expression of ViPS was assessed by slide agglutination test using a commercial Vi monoclonal antibody reagent (Statens Serum Institute Diagnostica A/S, Denmark; Lot 188L-8). Bacteria were washed twice in DPBS, and 100 μl of DPBS containing ~3×10⁴ CFUs was injected i.p. Three days post-infection, the liver and spleen were collected, and the tissues were processed using a Minilys tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Blood was collected into anti-coagulant and bacterial burden in the blood, and tissue homogenates were measured by counting CFUs on LB agar plates.

For each tumor sample, RNA extraction was performed with Trizol reagent (Invitrogen) after tissue dissociation using Minilys tissue homogenizer (Bertin, Ozyme). Total RNA was extracted from tumor using the Trizol method. Libraries were prepared from 1 μg of total RNA with the TruSeq Stranded Total RNA using Ribo-Zero (Illumina) following the manufacturer’s instructions. Once qualified, single-end libraries were sequenced using 1 × 76 bp output on a NextSeq 500 device (Illumina).
Paired-end transcriptome reads were pseudoaligned to the UCSC mm 10 reference genome and quantification of gene expressions as TPM (Transcript per Million) value were performed with the Kallisto algorithm [20 (link)]. The program was run with default options. Differential analysis was performed with DESeq2 R package [21 (link)] using log fold change shrinkage. A gene was considered significantly differentially expressed when the corresponding s-value < 0.005.
A gene set enrichment analysis was performed using the Cytoscape plug-in ClueGO [22 (link)] and the databases GO and KEGG 2018. The app was run using default parameters.

AsA contents were measured using a high-performance liquid chromatograph (HPLC) and electrochemical detector (Nihon Waters, Tokyo, Japan), as described previously^{35 (link)}. The gastrocnemius muscle was homogenized in 14 volumes of 5.4% metaphosphoric acid (MPA, Wako Pure Chemical, Osaka, Japan) using a Minilys Tissue Homogenizer three times (Bertin Technologies, Montigny-le-Bretonneux, France) at the maximum speed for 30 sec and centrifuged at 21,000 × g for 15 min at 4 °C. Supernatants obtained after centrifugation were suitably diluted, and samples were treated with Tris (2-carboxyethyl) phosphine hydrochloride and then incubated for 2 h at 4 °C to reduce dehydroascorbic acid to AsA to measure the total AsA (AsA plus dehydroascorbic acid is an oxidized form of AsA) levels. Thereafter, 5% MPA was added to the samples, which were centrifuged at 21,000 × g for 10 min at 4 °C. Then, the resulting supernatants were analyzed by HPLC using an Atlantis dC18 5 µm column (4.6 × 150 mm, Nihon Waters). The mobile phase consisted of 50 mM phosphate buffer (pH 2.8), 54 μM EDTA and 2% methanol at a flow rate of 1.3 mL/min, and electrical signals were recorded using an electrochemical detector with a glassy carbon electrode at +0.6 V.