Cryolys cooling unit

White skeletal muscle (∼100 mg) and vertebral bone tissues (∼100 mg) were homogenized with 1 ml of TRI Reagent Solution (Applied Biosystems, Alcobendas, Spain) using the Precellys^® Evolution Homogenizer coupled to a Cryolys cooling unit (Bertin Instruments, Montigny-le-Bretonneux, France). Next, RNA extraction was performed according to the manufacturer’s recommendations and resuspended in DEPC-treated water (RNase-free). RNA concentration of each sample was measured in a NanoDrop 2000 (Thermo Scientific, Alcobendas, Spain), and its integrity was confirmed in a 1% agarose gel (w/v) stained with 3% SYBR^®-Safe Gel Stainer (Bio-Rad, El Prat de Llobregat, Spain). Afterwards, 1,500 ng of RNA were treated with DNase I (Life Technologies, Alcobendas, Spain), to remove any residual genomic DNA, and retrotranscribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Sant Cugat del Vallès, Spain).

Metabolites were extracted using a modified Bligh–Dyer method, as described in Sinclair et al. [8 (link)]. Briefly, annelid tissue samples were first lysed at 6800 rpm (3 × 45 s), using a Precellys bead-mill attached to a Cryolys cooling unit (Bertin Technologies, Aix En Provence, France) that had been pre-chilled with liquid nitrogen. The samples then had 600 µL aliquot of methanol:chloroform (9:1 ratio) solution containing an internal standard (140 µM ¹³C5 -¹⁵N-Valine and 14 µM ¹³C6-Sorbitol) added to them) The tubes were then thoroughly mixed and centrifuged at 14,000 rpm at 0 °C for 15 min to generate upper (aqueous) and lower (organic) phases. A 400 µL sample of the upper phase was collected into a clean microcentrifuge tube. A 100 µL aliquot from each sample was taken for GC–MS analysis and a 10 µL aliquot was taken for LC–MS analysis.

For the determination of specific enzyme activity, cells were grown until stationary phase, washed once with 25 ml water, and resuspended in sodium phosphate buffer (100 mM, pH 7.0). Cell extract was made with glass beads (0.5 mm) and precellys 24 bead beater with a cryolys cooling unit (Bertin technologies, Aix-en-Provence Cedex, France). Total protein amount was determined by Bradford with bovine serum albumin (Fermentas UAB, Vilnius, Lithuania) as standard [37 (link)]. Reductase activity was assayed by following the oxidation of NAD (P) H at 340 nm using Ultrospec 2100pro spectrophotometer (Amersham Biosciences, Sweden). Data were collected using the software SWIFTII (Amersham Biosciences, Sweden). Samples were diluted so that the absorbance decreased linearly during 5 min. One unit of activity corresponds to 1 μmol NAD (P) H consumed per minute at 30°C. The assay contained sodium phosphate buffer (100 mM, pH 7.0), NAD (P) H (200 μM), ACP (10 mM) and cell extract.

To capture a broad spectrum of metabolites, we used the MxP^® Quant 500 kit (Biocrates Life Sciences AG, Innsbruck, Austria), capable of quantifying more than 600 metabolites from 26 compound classes, following the manufacturer’s instructions. All reagents, internal standards (IS), calibration standards, quality controls (QCs), and the test mix required for the MxP^® Quant 500 analysis were included in the kit. In addition, ACs were analyzed using the inhouse UHPLC-MS/MS-based AC assay from Biocrates, capable of separating different isomeric and isobaric groups of ACs. Isotope-labeled IS were used and the closest eluting deuterated IS were assigned for those ACs lacking their own isotope-labeled IS.
Samples were prepared the same way for both assays. Briefly, 50–80 mg of tumor tissues were transferred to 0.5 mL Precellys^® vials followed by the addition of 85:15 ethanol:0.01 M phosphate as a lysis buffer with a volume (µL) of three times the tissue weight (mg). Tissue homogenization was carried out at 4 °C and 5800 rpm using a Precellys^® 24 homogenizer coupled to a Cryolys^® cooling unit (Bertin Instruments, Bretonneux, France). The homogenates were centrifuged at 10,000 g for 2 min at 2–4 °C. Supernatants were collected and stored at −80 °C until analysis. Plasma samples as well as tissue homogenate supernatant were thawed on ice, then vortexed before use.

The concentration of total proteins in aorta homogenates was measured using a Pierce™ BCA Protein Assay Kit (23225; Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Aorta samples were homogenised automatically using Precellys Evolution combined with a Cryolys cooling unit (Bertin, Montigny-le-Bretonneux, France). After centrifugation (10,000× g, 10 min, 4 °C), the supernatant was analysed for total protein concentration.

Murine splenic B cells were purified by depletion using biotinylated antibodies (anti-CD43, anti-TER119, anti-CD11c, anti-TCRβ, anti-CD4, and anti-CD8) and streptavidin-conjugated beads (EasySep Streptavidin RapidSpheres) to greater than 95% purity and resuspended at 5 × 10⁷ cells per milliliter in cold PBS. B cells (100 µL) were added to Eppendorfs containing 900 µL prewarmed PBS and incubated for the indicated times in a 37 ^∘C heat block (or 900 µL cold PBS for ice conditions). Cells were pelleted at 400 relative centrifugal force (RCF) × 10 min at 4 ^∘C, and the pellets frozen in liquid nitrogen. Samples were kept at –80 ^∘C for a maximum of 12 h prior to sample preparation. Cell pellets were resuspended in 100 µL ice-cold methanol containing 0.1% formic acid and 10 nM 17:1 internal standard and homogenized using a Precellys 24 homogenizer with a Cryolys cooling unit (Bertin Technologies) and Precellys 0.5-mL soft tissue homogenizing tubes (Cayman Chemicals item 10428). Homogenization was conducted by two cycles for 15 s at 5,800 rpm (with a 30-s break) at a temperature lower than 4 ^∘C. After sample homogenization, 70 µL lysate was recovered and spun at 16,000 RCF and 40 µL of supernatant was transferred to glass vials with 9-mm autosampler inserts and immediately used for LC-MS/MS analysis.

Genomic DNA from sponge tissue (≈0.3 g) for 16S rRNA gene amplicon sequencing was extracted using the Precellys© Evolution homogenizer with a Cryolys cooling unit (Bertin Technologies, Montigny-Le-Bretonneux, France) and a DNeasy PowerSoil Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Extractions were performed using both internal and external sponge tissue in order to obtain the whole bacterial community. DNA concentrations were measured using a Nanoquant Spectrophotometer (Tecan, Switzerland). Approximate 500 ng of DNA sample were sent to the Dalhousie University CGEB-IMR¹ for V4–V5 rRNA gene library preparation and sequencing. Primers used correspond to 517f GTGYCAGCMGCCGCGGTAA and 926r CCGYCAATTYMTTTRAGTTT (Walters et al., 2015 (link)). Samples were multiplexed using a dual indexing approach and sequenced using an Illumina Miseq with paired-end 300 + 300 bp reads. All PCR procedures, primers, and Illumina sequencing detail were as described in Comeau et al. (2017) . Sequences were deposited at NCBI as BioProject with accession ID PRJNA541486.