A hydroxyproline assay kit (MAK008, Sigma, St. Louis, MO, USA) was used to detect the content of hydroxyproline in mouse right lung [26 (link)]. Briefly, the fresh lung tissue was homogenized in water (100 µL water of every 10 mg lung tissue), followed by hydrolyzing the samples after adding equivalent 12 N HCl for 3 h at 120 °C. Next, the samples were centrifuged (12,000 rpm, 4 °C, 3 min; Beckman Microfuge 20R) and extracted 10 µL supernatant of every sample, the samples were transferred to a 96-well plate and evaporated at 56 ℃. The samples in the plate were incubated for 5 min at 20 ℃ after added 100 µL chloramine T reagent into each well. Afterward, the samples were incubated for 90 min at 56 °C after mixed with 100 µL p-dimethylaminobenzaldehyde reagent. Absorbance of samples was measured by BioTek ELx800 plate reader at λ = 560 nm (Winooski, VT, USA). The hydroxyproline content of samples was calculated by a standard sample and presented as µg hydroxyproline per right lung.
Microfuge 20r
Manufactured by Beckman Coulter
Sourced in United States
The Microfuge 20R is a compact, high-speed refrigerated centrifuge designed for a wide range of laboratory applications. It can accommodate various tube sizes and features a temperature range of -10°C to 40°C. The Microfuge 20R provides reliable performance and consistent results for your research needs.
Automatically generated - may contain errors
Lab products found in correlation
Optima l 90k utracentrifuge, by Beckman Coulter (2 mentions)
Mps2 with dual heads, by Gerstel (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop onec spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Mak008, by Merck Group (1 mentions)
Gc fid 7890a, by Agilent Technologies (1 mentions)
4 methylvaleric acid, by Merck Group (1 mentions)
Model 5890 series 2, by Hewlett-Packard (1 mentions)
Nukol, by Merck Group (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Bile acid standards, by Merck Group (1 mentions)
Optima lc ms, by Thermo Fisher Scientific (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Mill q ultrapure water system, by Merck Group (1 mentions)
Pegrx ht, by Hampton Research (1 mentions)
Vivaspin 500, by Sartorius (1 mentions)
Freezone freeze dryer, by Labconco (1 mentions)
Acquity uplc xevo tq s, by Waters Corporation (1 mentions)
Ferric chloride, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
28 protocols using microfuge 20r
1
Hydroxyproline Quantification in Mouse Lungs
2
Short-chain Fatty Acid Quantification Protocol
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Aliquots (0.4 mL) from tubes were collected for SCFA analysis. Immediately after sampling, aliquots were mixed with 100 μL of the internal standard mixture (157.5 μL of 4-methylvaleric acid (nr 277827–5G, Sigma-Aldrich Inc., St. Louis, MO, USA), 1.47 mL of 85% phosphoric acid, 39 mg of copper sulfate pentahydrate) and the resulting blend was brought to 25 mL of final volume with purified water and 400 μL of copper sulphate solution (2.75 mg/mL) to halt fermentation. Samples were stored at −80 °C until analysis.
Defrosted samples were centrifuged at 3000× g for 10 min (Microfuge® 20R, Beckman Coulter, Brea, CA) and 4 μL was prepared for injection into a gas chromatograph (model 5890 Series II, Hewlett Packard, Palo Alto, CA, USA) equipped with a fused silica capillary column (NukolTM, Supelco nr 40369-03A, Bellefonte, PA, USA) and a flame ionization detector (GC-FID 7890A, Agilent Technologies, Inc., Santa Clara, CA, USA). SCFA was assayed and identified as previously described [36 (link)] using acetate, propionate and butyrate relative to 4-methyl valeric acid as standards (Supelco, Bellefonte, PA, USA).
Defrosted samples were centrifuged at 3000× g for 10 min (Microfuge® 20R, Beckman Coulter, Brea, CA) and 4 μL was prepared for injection into a gas chromatograph (model 5890 Series II, Hewlett Packard, Palo Alto, CA, USA) equipped with a fused silica capillary column (NukolTM, Supelco nr 40369-03A, Bellefonte, PA, USA) and a flame ionization detector (GC-FID 7890A, Agilent Technologies, Inc., Santa Clara, CA, USA). SCFA was assayed and identified as previously described [36 (link)] using acetate, propionate and butyrate relative to 4-methyl valeric acid as standards (Supelco, Bellefonte, PA, USA).
3
Quantification of Hippocampal FMN Levels
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FMN concentrations in the hippocampus after intraperitoneal delivery of free FMN, and intravenous delivery of PBS, free FMN, or MNPs@FMN were examined using UPLC‐MS/MS analysis. Briefly, each tissue sample (≈15 mg) was mixed with 20 µL of deionized water and homogenized for 3 min, and 150 µL of methanol containing internal standard was added to extract the metabolites. The sample was homogenized for another 3 min and then centrifuged at 18 000 × g for 20 min (Microfuge 20R, Beckman Coulter, Inc., Indiannapolis, IN, USA). Then the supernatant was transferred to a 96‐well plate and subjected to a UPLC‐MS/MS system (Acquity UPLC with Xevo TQ‐S Mass Spectrometer, Waters Corp., Milford, MA, USA). Samples were injected onto an Atlantis Premier analytical column (100 × 2.1 mm, 1.7 µm) at a flow rate of 0.4 mL min−1. The Q Exactive series mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3 kV. The raw data files generated by the UPLC‐MS/MS were processed using MassLynx software (v4.1, Waters, Milford, MA, USA) to perform peak alignment, peak picking, and quantitation for FMN.
4
Comprehensive Bile Acid Quantification
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Bile acid standards (Steraloids, USA), six stable isotopes labeled standards (C/D/NIsotopes, Canada/Steraloids), ammonium acetate (analytical grade, Sigma–Aldrich, St Louis, MO, USA), methanol (Optima LC-MS), acetonitrile (Optima LC-MS), isopropanol (Optima LC-MS), glacial acetic acid and formic acid (Optima LC-MS) were all purchased from ThermoFisher Scientific (Fairlawn, NJ, USA). The following equipment was used: ACQUITYUPLC-XevoTQ-S liquid-mass spectrometer (WatersCorp., Milford, MA, USA); Mill-Q ultrapure water system (Millipore, Billerica, MA, USA); homogenizer (BB24; NextAdvance, Averill Park, NY, USA); microcentrifuge (Microfuge20R; Beckman Coulter, Indianapolis, IN, USA); and lyophilizer (Labconco, Kansas City, MO, USA).
5
Crystallization of Thrombin-Aptamer Complexes
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For crystallization experiments, the thrombin-aptamer complexes were prepared following a standard protocol (32–34 (link),38 (link),47 (link),48 (link)), using the PPACK-inhibited thrombin. In order to hamper the formation of highly stable intermolecular aptamer species, the complexes were prepared in the presence of sodium ions (25 mM sodium phosphate buffer pH 7.4, 100 mM NaCl). The complexes were concentrated to about 9 mg ml−1 using 10 kDa-cutoff Centricon mini-concentrator (Vivaspin 500, Sartorius) and a refrigerated centrifuge (Microfuge 20R - Beckman Coulter).
An initial extensive screening of sitting-drop crystallization experiments at 20 and 4°C in 96-well plates was carried out using a Mosquito Crystal protein crystallization robot (SPT Labtech) and commercially available crystallization screens (NeXtal QIAGEN Classics and Classics II; Jena Bioscience JBScreen PACT++; Hampton Research PEGRx HT).
The optimization of the starting conditions was performed by hanging drop vapour diffusion method mixing 0.5 μl complex solution with 0.5 μl reservoir solution at 20°C. Crystals suitable for X-ray diffraction data collection grew only for thrombin-M08s-1_41mer complex (see results), in 20% w/v PEG 8000, 0.2 M magnesium acetate and 0.1 M sodium cacodylate pH 6.5.
An initial extensive screening of sitting-drop crystallization experiments at 20 and 4°C in 96-well plates was carried out using a Mosquito Crystal protein crystallization robot (SPT Labtech) and commercially available crystallization screens (NeXtal QIAGEN Classics and Classics II; Jena Bioscience JBScreen PACT++; Hampton Research PEGRx HT).
The optimization of the starting conditions was performed by hanging drop vapour diffusion method mixing 0.5 μl complex solution with 0.5 μl reservoir solution at 20°C. Crystals suitable for X-ray diffraction data collection grew only for thrombin-M08s-1_41mer complex (see results), in 20% w/v PEG 8000, 0.2 M magnesium acetate and 0.1 M sodium cacodylate pH 6.5.
6
Quantification of Bile Acids in Liver Tissue
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A total of 10 mg liver tissue was cut and transferred to a microcentrifuge tube. Also, 25 mg beads and 20 μl ultrapure water were mixed using a homogenizer (BB24, Next Advance, Inc., Averill Park, NY, USA). Samples were mixed with 180 μl acetonitrile/methanol (8:2) containing 10 internal standards and centrifuged at 13000 rpm for 20 min and 4 °C (Microfuge 20R, Beckman Coulter, Inc., Indianapolis, IN, USA). Following centrifugation, the supernatant was transferred to a 96-well plate and freeze-dried with FreeZone freeze dryer (Labconco, Kansas City, MO, USA). The dried sample powder and freeze-dried standard were remixed with 1:1 (v/v) acetonitrile/methanol (80/20, v/v) and ultrapure water, and then centrifuged at 13000 rpm for 20 min and 4 °C (Microfuge 20R). A total of 5 μl supernatant was transferred to a 96-well plate and then subjected to LC-MS analysis.
Bile acids were analyzed using an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA), in accordance with previous reports [26 (link), 27 (link)].
Bile acids were analyzed using an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA), in accordance with previous reports [26 (link), 27 (link)].
7
Reducing Power Assay for Kiwiberry
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The reducing power assay was performed as described by Oyaizu [99 (link)]. A mixture of 300 μL of kiwiberry extract, 300 μL of 200 mM sodium phosphate buffer (pH 6.6), and 300 μL of 1% (w/v) potassium ferricyanide (Sigma-Aldrich) was incubated for 20 min at 50 °C in a water bath (JSWB-22(T); JSR, Korea). The reactants were mixed with 300 μL of 10% (w/v) trichloroacetic acid (Sigma-Aldrich) solution and then centrifuged at 13,000× g using a microcentrifuge (microfuge 20R; Beckman Coulter). Subsequently, 300 μL of 0.1% (w/v) ferric chloride (Sigma-Aldrich) was added to 300 μL of the supernatant, and the absorbance of the solution was measured at 700 nm using a multi-mode microplate reader.
8
Extracellular Vesicle Isolation Protocol
9
Untargeted Metabolomics Profiling of Plasma
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The untargeted metabolomics profiling was implemented on XploreMET platform (Metabo-Profile, Shanghai, China). The sample preparation was conducted as their published methods with minor modifications [24 , 25 (link)]. In brief, the plasma samples were centrifuged at 3000×g and 4 °C for 5 min (Microfuge 20R, Beckman Coulter, Inc., Indianapolis, IN, USA) after thawing to separate debris or a lipid layer. Metabolites were extracted from plasma samples (50 μL) with 10 μL of internal standard (0.5 mM 4-Chlorophenylalanine) and 175 μL of pre-chilled methanol: chloroform (3:1) followed by centrifugation at 14, 000×g for 20 min at 4 °C. Then each 200 μL of the supernatant was transferred into an autosampler vial (Agilent Technologies, Foster City, CA, USA). The resting supernatant from each sample was pooled to prepare quality control samples. Following solvent evaporation and lyophilization, the dried samples were derivatized with 50 μL of methoxyamine (20 mg/ml in pyridine) for 2 h, followed by silylanization with 50 μL of MSTFA (1% TMCS) for 1 h prior to injection. Above two steps were performed by a robotic multipurpose sample MPS2 with dual heads (Gerstel, Muehlheim, Germany).
10
Extracellular Vesicle Isolation Protocol
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After BALF was centrifuged at 300-500 g for 10 minutes to remove cells, fluid was serially centrifuged at 1,200 g for 20min, 10,000 g for 30min and 100,000 g for 2hrs all at 4oC in a Microfuge 20R (Beckman Coulter) and an Optima L-90K Utracentrifuge (Beckman Coulter). For density gradient flotation experiments, the 100,000 g pellet was collected in a 60% sucrose solution followed by layering of 40% and 20% sucrose. Samples were spun overnight (16-18hrs) at 150,000 g then particles recovered from three fractions by centrifugation at 150,000 g for 1hr. All fractions were finally resuspended in PBS.
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