Speedvac spd121p

After disruption of the cellular pellets, as described for LC-MS (liquid chromatography-mass spectrometry), 150 μL of the supernatant were transferred into a new tube and evaporated to dryness in a SpeedVac SPD121P (Thermo Fisher Scientific, Waltham, MA) at 35°C. The metabolite extracts were re-suspended in 150 μL MilliQ water, centrifuged (15700 × g, 15 min, 4°C) and injected into the instrument.

Pellets with 5 × 10⁶ uninfected or infected macrophages were re-suspended in 350 µL of cold methanol/water (3:1, v:v) and 25 mg of glass beads (710–1180 µM, G1152, SigmaAldrich, Germany), followed by four cycles of frost/defrosting in a liquid N₂/37 °C bath. The cells were disrupted at 50 mHz for 10 min in TissueLyser LT (Qiagen, Germany). The samples were clarified by 15,700× g for 10 min at 4 °C centrifugation and the supernatant was collected and evaporated to dryness by SpeedVac SPD121P (Thermo Fisher Scientific, Waltham, MA.) at 35 °C for 2 h. After this, the solid residue was re-suspended in 60 µL 0.1 M formic acid with 0.2 mM of methionine sulfone, homogenized for 15 min in a vortex, and centrifuged at 15,700× g for 15 min at 4 °C. The 35 µL of supernatant was transferred to polypropylene vials (Agilent Techno Vials, Waldbronn, Germany) for analysis. Quality-control (QC) samples were prepared by pooling equal volumes of all the samples and they were analyzed along the analytical sequence, to evaluate the stability and performance of the instruments during measurements [27 (link)].

Bacteria were cultured in 120 mL of GYM broth for four weeks, under shaking (120 rpm) at 28 °C. Cultures were then centrifuged at 3000 rpm for 10 min and biomass and supernatants were separated. For the preparation of methanol extracts, biomass was suspended (w/v) with two volumes of methanol, homogenized, and sonicated for five minutes, after which it was stirred for 48 h at 120 rpm at 25 °C in darkness [24 ]. To prepare ethyl acetate extracts, 50 mL of the supernatant was placed in a separatory funnel with 100 mL of the solvent, which was manually shaken for five minutes and the organic phase was discarded [25 (link)]. Solvents were removed with a SpeedVac SPD121P concentrator (Thermo Scientific, San Jose, CA, USA) at 35 °C. Then, 1 mg of solvent-based extracts was suspended in 20 µL of 99.5% dimethylsufoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), filtered by 0.20 µm filters (Corning Incorporated, Corning, NY, USA), and stored at −20 °C, until use [26 (link)]. The final DMSO concentration in cell cultures was less than 1%, which did not affect cell viability. Extract yields were analyzed by the following Formula (1):

The samples were derived from 12 different Italian fig cultivars collected in September 2013 from an experimental plot located in Carmignano (Tuscany, Italy). The overall information of all analyzed fig cultivars is reported in Table 3. The fig fruits were manually peeled, and peel was then freeze-dried (Heto Hetovac VR-1 and CT 110 Vacuum Concentrator, Heto Lab Equipment, Roskilde, Denmark) and subsequently homogenized using a mortar. All samples were then stored at −80% until (poly)phenol extraction. Dried fig (poly)phenols were extracted by accurately weighing 200 mg of freeze-dried powder in a plastic tube and by adding 5 mL of a solvent mixture containing acetone/water/formic acid, 80/19.5/0.5 (v/v/v) [10 (link)]. Tubes were vortexed for approximately 30 s, sonicated in an ultrasonic bath for 10 min, vortexed again for 30 s and finally centrifuged for 10 min at 2575 g. The supernatant was then evaporated through a centrifugal concentrator (SpeedVac SPD121P, Thermo Fisher Scientific Inc., San José, CA, USA) and the residual pellet was resuspended in 200 μL of 0.1% aqueous formic acid before uHPLC-MSⁿ analyses.

Frozen root tissue was homogenized to a fine powder in liquid nitrogen using a mortar and pestle. 150 mg of this powder was transferred to a 2 ml safe-lock Eppendorf micro-extraction tube. Samples were extracted with 1.5 ml of 60% acetone/water (v/v), containing 2-mm yttria stabilized zirconium oxide beads (Next Advance, Inc., Troy, NY, USA) and 5 pmol of internal standards (GR24, [²H₆]-2′-epi-5-DS, [²H₆]-5-DS) in order to control for the extraction recovery. Additional homogenization was performed using a bead mill—Tissue-Lyzer II (Qiagen Sciences, Inc., Germantown, MD, USA) at 27 Hz for 3 min. Subsequently, samples were sonicated for 3 min in a Brandson 3510 ultrasonic bath under cooling with ice water and shaken for 30 min in a cold room (4 °C) using a Loopster benchtop laboratory rotator (IKA^®-Werke GmbH & Co. KG, Staufen, Germany) at 20 rpm. After centrifugation (16.000 rpm/10 min/4 °C), the supernatant was collected and the organic solvent was removed in vacuo (Thermo Fisher Scientific SPD121P SpeedVac, Whaltman, MA, USA). The remaining aqueous phase (approx. 0.9 ml) was made up to 1.5 ml by addition of 25% acetonitrile/water (v/v) in order to quantitatively dissolve SL analytes and achieve suitable SPE loading conditions (5–10% acetonitrile/water, v/v).

Total protein content was determined using the bicinchoninic acid (BCA) method previously described [81] (link) with slight modifications. Briefly, the pellet from 50 worms was dried in a Speed Vac Concentrator (SPD12 1P SpeedVac, Thermo Scientific), 20 µl of 1 M NaOH was added to the dry pellet. Fat was degraded by heating at 70°C for 25 min and 180 µl of distilled water was added. After vortexing, the tubes were centrifuged at 14000 rpm for 5 min and 25 µl of the supernatant were transferred into a 96 well plate. Next, 200 µl of the BCA reagent prepared according manufacturer's instructions (Pierce BCA Protein Assay Kit, Thermo Scientific) and added to the sample. After incubation at 37°C for 30 min, the plate was cooled to room temperature and absorbance was measured using the POLARstar Omega luminometer (BGM Labtech) at 560 nm.