Plasma was collected from mice and used directly for the activity assays. Tissues were homogenized using a Benchmark BeadBlaster Homogenizer in ice-cold PBS, centrifuged to remove debris (5 min at 1000 x g), and the supernatant was collected and used for activity assays. For assays using liver membranes, total liver homogenates were transferred into ultracentrifuge inserts and spun at 100,000 x g on a Beckman Centrifuge I8-70M for 1 hr at 4°C. In vitro enzymatic reactions were conducted in glass vials and initiated by the addition of 100 µg protein. Final reaction conditions for the hydrolase reactions were 100 µM substrate (C20:4-Gly or C20:4-Phe) and 100 µg protein in 100 µl PBS, and for the synthase reactions were 1 mM Phe, 1 mM oleic acid, and 100 µg protein in 100 µl PBS. After 1 hr at 37°C, reactions were quenched with 400 µl 2:1 v/v acetonitrile:methanol and vortexed. Reaction vials were centrifuged at 2000 x g to remove debris, and the supernatant was collected and analyzed by LC-MS as described below. For inhibitor assays, tissue lysates were treated with the indicated inhibitors for 10 min at room temperature before the introduction of the indicated substrates.
Beadblaster homogenizer
Manufactured by Benchmark Scientific
Sourced in United States
The BeadBlaster homogenizer is a device designed for efficient homogenization of a wide range of sample types. It utilizes high-speed bead-beating technology to effectively disrupt cells and tissues, facilitating the extraction of biomolecules such as proteins, nucleic acids, and other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Thermo Fisher Scientific (2 mentions)
Speedvac, by Thermo Fisher Scientific (2 mentions)
Optima lc ms grade water, by Thermo Fisher Scientific (2 mentions)
Disruption beads, by Research Products International (2 mentions)
Metabolomics amino acid mix standard, by Cambridge Isotopes (2 mentions)
Mobio powersoil dna isolation kit, by Qiagen (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Leupeptin, by Roche (1 mentions)
Aprotinin, by Roche (1 mentions)
Formic acid, by Merck Group (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
9 protocols using beadblaster homogenizer
1
Enzymatic Activity Assays of Lipid Metabolites
2
Quantifying Metabolites in Plasma and Tissues
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Frozen plasma (30 μL) was extracted in 160 μL of 1:1 (vol/vol) acetonitrile:methanol. Tissues were extracted in 500 μL 2:2:1 (vol/vol/vol) acetonitrile:methanol:water on a BeadBlaster homogenizer (Benchmark Scientific, Inc.) for 1 min. In both cases, 100 nmol of C15:0-Phe was included as an internal standard for absolute quantitation. Extracts were centrifuged at 5,000 × g for 10 min to remove debris. The supernatant was isolated and centrifuged again at 5,000 × g for 10 min. Finally, the twice-clarified supernatant was transferred to a mass spectrometry vial and analyzed by LC-MS as described in SI Appendix, SI Experimental Procedures .
3
Soil and Compost Nematode DNA Extraction
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Genomic DNA was isolated from compost exposed nematodes (~ 700) or soil (0.25 g) using the MO BIO PowerSoil DNA Isolation Kit (12,888; MO BIO Laboratories; Carlsbad, CA, USA), with slight adjustments. For homogenization and cell lysis, we attached the MO BIO kit’s PowerBead Tubes to the Benchmark Scientific BeadBlaster Homogenizer (D1030-E; Benchmark Scientific; South Plainfield, NJ, USA) and homogenized and lysed cells for 60 s at 2800 rpm. Final gDNA was released from the silica membrane using 40 μL sterile, nuclease-free water (Promega; Madison, WI, USA).
4
Cytokine and Chemokine Analysis of Tumor Samples
5
Metabolite Extraction and Sample Preparation
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Prior to extraction, samples were moved from −80°C storage to wet ice and thawed. Extraction buffer, consisting of 80% methanol (Cat# 615130025, Thermo Fisher) and 500 nM metabolomics amino acid mix standard (Cat# MSK-A2–1.2, Cambridge Isotope Laboratories), was prepared and placed on dry ice. Samples were extracted by mixing 50 μL of sample with 950 μL of extraction buffer in 2.0 mL screw cap vials containing ~100 μL of disruption beads (Cat# 9835, Research Products International). Each sample was homogenized for 10 cycles on a BeadBlaster homogenizer (Benchmark Scientific). Cycling consisted of a 30 s homogenization time at 6 m/s followed by a 30 s pause. Samples were subsequently spun at 21,000 rcf for 3 min at 4°C. A set volume of each (450 μL) was transferred to a 1.5 mL tube and dried down by SpeedVac (Thermo Fisher). Samples were reconstituted in 50 μL of Optima LC/MS grade water (Cat# W6500, Fisher Scientific). Samples were sonicated for 2 min, then spun at 21,000 rcf for 3 min at 4°C. Twenty μL were transferred to LC vials containing glass inserts for analysis. The remaining sample was placed in −80°C for long term storage.
6
Evaluation of MERS-CoV Vaccine Efficacy
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The K18-hDPP4 mice (n = 13) were i.n. immunized with the EMC2012-CA 22 °C vaccine virus (2 × 104 pfu) and i.n. inoculated with Korean MERS-CoV (2 × 104 pfu) 4 weeks after the vaccination. As controls, mice (n = 13) vaccinated with PBS and infected with MERS-CoV virus were used. Before the immunized mice were challenged with wild-type MERS-CoV (Korean MERS-CoV/2015), their sera were collected to measure the titers of the viral neutralizing antibody. The body weight changes and mortality of the challenged mice were recorded. Six days after challenge, three mice per group were euthanized to measure the virus titers in their nasal turbinate, brain, lung, kidney, and stained tissues (brain, lung, and kidney). Each tissue sample weighing 0.1 g was homogenized in 1 mL of PBS (pH 7.4) using a BeadBlaster homogenizer (Benchmark Scientific, Edison, NJ, USA). The viral titers were measured using log10TCID50/g.
7
Metabolomics Extraction and Sample Prep
8
MERS-CoV Infection in Transgenic Mice
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The K18-hDPP4 mice (n = 13 per group) were anaesthetized with isoflurane USP (Gujarat, India) and were then intranasally (i.n.) infected with 50 μL (2 × 104 pfu) of the cold-adapted MERS-CoV vaccine virus, EMC2012-CA 22 °C, or wild-type MERS-CoV virus, EMC2012. Mice infected with PBS were used as controls. Following infection, the mice were closely observed for body weight changes and death. On day 6 post-infection (p.i.), the mice (n = 3 per group) were euthanized. Nasal turbinates, brain, lungs, and kidneys were collected as tissue samples for subsequent analysis. Each tissue sample weighing 0.1 g was homogenized in 1 mL of PBS (pH 7.4) using a BeadBlaster homogenizer (Benchmark Scientific, Edison, NJ, USA). The resulting homogenates were used to determine the viral titers, expressed as log10TCID50/g. The remaining tissue samples were reserved for histopathological examination.
9
Plant DNA Extraction and Quantification
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Total genomic DNA was extracted from fresh plant material using a DNeasy TM Plant Mini Kit (Qiagen, UK). For each sample, genomic DNA was extracted from 100 mg frozen leaf tissue, which was first ground using a BeadBlaster homogenizer (Benchmark Scientific, USA). Extracted DNA was quantified by means of a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., USA) and visualized on 1% agarose gel stained with ethidium bromide.
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