50±5 mg hippocampal tissue (Study 3) were extracted in 800 μl of cold 50:50 (v/v) choloroform:methanol containing a mixture of isotopically labeled internal standards in methanol as previously described [20] (link). Two extraction blanks (no sample) were also included. LC-MS grade water (400 μl) was added to initiate phase separation, samples were vortexed and centrifuged (2400×g, 15 min). 200 μl of the polar (methanol:water) phase from each sample (individual animals) was collected and quality control standards also prepared by pooling 200 μl from each sample. Samples and standards were dried using a Savant Speedvac centrifugal concentrator (ThermoFisher Scientific, UK) and stored at 4 °C.
Savant speedvac
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, Italy
The Savant SpeedVac is a vacuum concentrator designed for the rapid evaporation of solvents from liquid samples. It utilizes a combination of vacuum and temperature control to efficiently remove solvents, leaving behind the desired sample components.
Automatically generated - may contain errors
Lab products found in correlation
Sequencing grade modified trypsin, by Promega (2 mentions)
Isotype control antibody, by BioXCell (2 mentions)
αpd 1 antibody, by BioXCell (2 mentions)
Matrigel, by Corning (2 mentions)
Thermomixer, by Eppendorf (2 mentions)
Zirconia silica bead, by Biospec (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Waters Corporation (1 mentions)
Sep pak c18 cartridge, by Waters Corporation (1 mentions)
Zymoclean large fragment dna recovery kit, by Zymo Research (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Aqueous acetonitrile, by Avantor (1 mentions)
Agilent 1200 series system, by Agilent Technologies (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Techne, by Cole-Parmer (1 mentions)
Synergi hydro rp column, by Phenomenex (1 mentions)
Qtrap 5500 ms, by AB Sciex (1 mentions)
Infinity 1260 lc, by AB Sciex (1 mentions)
Bioprime array cgh genomic labeling system, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
79 protocols using savant speedvac
1
Hippocampal Metabolite Extraction Protocol
2
Quantitative Biofilm Cultivation and Analysis
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A. oris biofilms were cultivated according to a previously published protocol with some modification (21 (link)). Overnight cultures of A. oris strains were used to inoculate fresh cultures (1:100 dilution) in HIB supplemented with 1% sucrose in 24-well plates, which were allowed to grow for 48 h at 37 °C in the presence of 5% CO2. Biofilms were washed with phosphate buffered saline (PBS) three times prior to drying in a Savant speedvac (Thermo Scientific). Biofilms were stained with 1% crystal violet for 10 min, washed 3–5 times with water, de-stained, and dissolved in 30% acetic acid for 5 min, and quantified by measuring absorbance at 580 nm.
3
Proteomic Sample Preparation for Mass Spectrometry
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Enriched proteins were denatured with 4 M guanidine hydrochloride (GuHCl), and disulfide bonds were reduced with 5 mM 1,4-dithiothreitol (DTT) at 65°C for 1 h. Then, 15 mM IAA was added and incubated at room temperature (in the dark) for 1 h. Samples were incubated with 10 mM DTT for 15 min at room temperature, and proteins were precipitated with 8 volumes of cold acetone and 1 volume of cold methanol for 3 h at -80°C and centrifuged at 14,000 × g for 10 min at 4°C. The supernatant was discarded, and the precipitate was washed (2 times) with cold methanol and dried in a SpeedVac (Savant SpeedVac, Thermo Fisher Scientific Inc., Asheville, NC, United States). Proteins were dissolved in 10 μL 100 mM NaOH, 15 μL H2O2 and 75 μL of 50 mM HEPES buffer (pH 7.5); subsequently, 2 μg of sequencing-grade modified trypsin (Promega, Madison, WI, United States) was added to samples and incubated overnight at 37°C (Kleifeld et al., 2011 (link); Castilho et al., 2014 (link)). Samples were diluted up to 500 μL of 0.1% trifluoroacetic acid (TFA) for trypsin inactivation and loaded into a solid-phase extraction Sep-Pak C18 cartridge (Waters, Milford, MA, United States) according to the protocol (Menin et al., 2008 (link)). After desalting, samples were dried in SpeedVac and resuspended in 0.1% formic acid prior to nanoliquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
4
Peptide Desalting Using C18 STAGE Tips
5
Bilberry Polyphenol Extraction and Purification
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Extraction, enrichment, and hydrolysis of the
bilberry were carried out using essentially the same method as described
previously.15 (link) Briefly, the enriched bilberry
extract powder was extracted with 75% aqueous ethanol containing 0.1%
HCl and enriched by loading the concentrated extracts on an XAD-761/Diaion
HP-20 (1:1) column. The polyphenols, including anthocyanins, were
eluted with methanol. Pooled elutes were concentrated and hydrolyzed
with 2 N HCl (∼5 volumes). Hydrolysates were purified using
C18 Sep-Pak cartridges (Waters, Milford, MA, USA). Anthocyanidins
and other polyphenols were eluted with acidified (0.01% HCl) methanol.
The enriched extracts were dried under reduced pressure using a Savant
Speed-Vac (Thermo Scientific, USA) and stored at −20 °C
until use. The enriched extracts were dissolved in acidified water,
and anthocyanidins were selectively extracted in isoamyl alcohol and
dried under vacuum.16 (link) The extracted anthocyanidins
were further purified by loading on the C18 cartridges per the manufacturer’s
guidelines.
bilberry were carried out using essentially the same method as described
previously.15 (link) Briefly, the enriched bilberry
extract powder was extracted with 75% aqueous ethanol containing 0.1%
HCl and enriched by loading the concentrated extracts on an XAD-761/Diaion
HP-20 (1:1) column. The polyphenols, including anthocyanins, were
eluted with methanol. Pooled elutes were concentrated and hydrolyzed
with 2 N HCl (∼5 volumes). Hydrolysates were purified using
C18 Sep-Pak cartridges (Waters, Milford, MA, USA). Anthocyanidins
and other polyphenols were eluted with acidified (0.01% HCl) methanol.
The enriched extracts were dried under reduced pressure using a Savant
Speed-Vac (Thermo Scientific, USA) and stored at −20 °C
until use. The enriched extracts were dissolved in acidified water,
and anthocyanidins were selectively extracted in isoamyl alcohol and
dried under vacuum.16 (link) The extracted anthocyanidins
were further purified by loading on the C18 cartridges per the manufacturer’s
guidelines.
6
Comparative Genomics of Anabolic and Catabolic Morphs
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DNA was extracted separately from 51 ana-morph and 63 cata-morph individuals using the Qiagen DNeasy Plant Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The DNA was quantified using the Qubit dsDNA BR Assay kit on the Qubit 2.0 Fluorometer. Two pools of the respective ana- and cata-morphs were prepared, with equimolar quantities of each individual sample. Low-molecular-weight DNA fragments were removed from the DNA pools by gel extraction using the Zymoclean Large Fragment DNA Recovery kit (Zymo Research, Irvine, CA, USA). The DNA was then concentrated using the Savant SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA) to reach a suitable final concentration. The samples were sequenced by Novogene (UK) with 150 bp paired-end reads on an Illumina NovaSeq 6000 instrument (Illumina, San Diego, CA, USA), aiming at generating 50x sequencing coverage per pool.
7
Intracellular Nucleotide Quantification in RAW264.7 Cells
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Following exposure to sodium acetate, RAW264.7 cells were washed three times with ice-cold PBS, and intracellular nucleotides were extracted by adding 3 mL of ice-cold aqueous acetonitrile (50%, v/v) (VWR, Radnor, PA, USA) to cells. The resulting suspension was maintained on ice for 10 min, followed by centrifugation at 14,000× g for 1 min at 0 °C. Next, the supernatant was collected and dried using a refrigerated Savant SpeedVac vacuum concentrator (Thermo Fisher Scientific, Waltham, MA USA), after which the dried extract was resuspended in 240 μL of deionized water and filtered using a 0.22 μm syringe filter unit for high-performance liquid chromatography (HPLC) analysis [45 (link)]. The chromatographic separation and analysis were performed on an Agilent system (1200 series) equipped with a diode-array detector and a C18 reverse-phase column (Kromasil, 5 μm, 100 Å; 4.6 × 150 mm) at a flow rate of 1 mL/min and a linear gradient of acetonitrile (0~7%) in 10 mM triethylammonium acetate buffer (Glen Research, Sterling, VA, USA) over 20 min. AMP and ATP were identified based on their retention times.
8
Subcutaneous Tumor Xenograft Assay
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Subcutaneous tumors were initiated by injecting tumor cells in 50% Matrigel (Corning, Bedford, MA) into the subcutaneous space on the left and right flanks of mice. Two × 105 murine PDAC cells or 5 × 105 human PDAC cells were used for each tumor. In vivo G-1 treatments were performed by first dissolving G-1, synthesized as described previously,9 in 100% ethanol at a concentration of 1 mg/mL. The desired amount of G-1 was then mixed with an appropriate volume of sesame oil, and the ethanol was evaporated off using a Savant Speed Vac (Thermo Fisher Scientific, Waltham, MA), leaving the desired amount of G-1 dissolved in 50 μL sesame oil per injection at a 10 mg/kg dose. Vehicle injections were prepared in an identical manner using 100% ethanol. Vehicle and G-1 injections were delivered through subcutaneous injection as indicated in each experimental timeline. Isotype control antibody (Clone: 2A3; BioXcell, West Lebanon, NH) and αPD-1 antibody (Clone: RMP1-14; BioXcell) were diluted in sterile phosphate-buffered saline (PBS) and delivered through intraperitoneal injections at a dose of 10 mg/kg.
9
Exosomal Protein Analysis by Western Blot
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Intact exosomes (100 μL) were dried in a Savant SpeedVac (Thermo Fisher Scientific), dissolved in 20 μL of sodium dodecylsulfate (SDS) sample loading buffer (EzApply; ATTO, Tokyo, Japan), and boiled for 5 min at 95 °C. Proteins were separated in 10–20% SDS-PAGE gels (e-PAGEL; ATTO, Tokyo, Japan) and transferred to PVDF membranes (ATTO), which were blocked with PVDF Blocking Reagent for Can Get Signal® (TOYOBO Biotech support Department, Osaka, Japan) for 1 h at room temperature (RT; approximately 20 °C). Immunodetection was performed by incubation with anti-CD63 (H-193) rabbit antibody (dilution 1:600; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 60 min at RT and then with horseradish peroxidase-conjugated anti-rabbit IgG (dilution 1:2000; Cell Signaling Technology Japan, Tokyo, Japan) for 60 min at RT. Signals were generated using an ECL kit (GE Healthcare, Tokyo, Japan) according to the manufacturer’s protocol.
10
In-Gel Protein Digestion Protocol
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In-gel digestion was performed according to Shevchenko and co-workers [31 (link)] and modified as specified below. Protein spots were washed four times for 15 min in destaining solution (40% Ethanol/50 mM NH4HCO3), reduced with 65 mM DTT in 50 mM NH4HCO3 for 20 min at 600 rpm, and alkylated with 260 mM Iodoacetamide in 50 mM NH4HCO3 for 20 min at 600 rpm using thermomixer (Eppendorf, Hamburg, Germany). The gel plugs were dehydrated with 100% acetonitrile (ACN), and vacuum dried using Savant Speed Vac® (Thermo Fisher Scientific, Schwerte, Germany), followed by rehydration in 25 mM NH4HCO3 containing 75 ng/μl of trypsin (Trypsin from porcine pancreas, Proteomics Grade, Sigma-Aldrich, St. Louis, USA). Digestion was carried out for 3 h at 37°C and continued over night after addition of elution buffer (25 mM NH4HCO3/10%ACN), in thermal cycler (Techne by Bibby Scientific, Staffordshire, UK). The digestion was stopped by addition of 5% formic acid (FA) to a volume of 10% of the total volume. The samples were stored at -80°C until further analysis.
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