Thirty millilitres of log-phase mycobacterial culture was centrifuged for 5 min at 2,000g at room temperature. Supernatant was removed and filtered twice with 0.22 µm filter. The pellet was washed with wash buffer (PBS–Tween-80 0.05% or PBS–Tyloxapol 0.05%), then with PBS. The pellet was resuspended in 500 µl of PBS containing protease inhibitor and transferred to a 2 ml screw cap tube 1/3 full of glass beads (Sigma, G-1145). Samples were ribolysed at setting 6.5 for 30 s, then placed on ice for 5 min and centrifuged twice at 4,000g for 1 min at 4 °C. The remaining supernatant was centrifuged at 4,000g for 10 min at 4 °C and 500 μl transferred to a Millipore Ultrafree-MC centrifugal filter device. Samples were filtered two times Millipore Ultrafree-MC centrifugal filter device at 4,000g for 5 min at 4 °C.
Ultrafree mc centrifugal filter device
Manufactured by Merck Group
Sourced in United States
The Ultrafree-MC Centrifugal Filter Devices are laboratory equipment used for the rapid separation and concentration of samples. They utilize a centrifugal force to efficiently filter and concentrate a wide range of materials, including proteins, nucleic acids, and other macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Exoquick exosome precipitation solution, by System Biosciences (2 mentions)
Type 45 ti rotor, by Beckman Coulter (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Centrifuge 5415r, by Eppendorf (1 mentions)
N dodecyl β d maltopyranoside, by Merck Group (1 mentions)
Lc ms grade methanol, by Avantor (1 mentions)
Ammonium formate, by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Yuanye Bio-Technology (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Yuanye Bio-Technology (1 mentions)
7 protocols using ultrafree mc centrifugal filter device
1
Mycobacterial Cell Lysis and Protein Extraction
2
Cytokine Profiling in Tissue Homogenates
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Tissue for cytokine analysis was weighed and transferred to individual 3.6 mL CryoTubes containing Tissue Homogenate Lysis Buffer (Ampliqon, Skovlunde, Denmark). The buffer was a solution of 200 mM NaCl, 5 mM EDTA, 10 mM Tris, 10% glycerin, 1 mM PMSF, 1 μg mL−1 leupeptin, and 28 μg mL−1 aprotinin (pH 7.4). The buffer was kept cold (approx. 4°C) at all times. The tubes were immediately snap-frozen in liquid nitrogen and stored at −80°C until homogenized. Using an Ultra-Turrax T25 basic disperser (IKA-Werke, Staufen, Germany) the colon segments were homogenized and the homogenates were centrifuged three times for 15 min at 10,000 ×g and 4°C, twice in Eppendorf tubes and the last time in an Ultrafree MC-Centrifugal Filter device, 5 μm pore size (Millipore, Billerica, Massachusetts, USA). The supernatants were analyzed for levels of colonic cytokines and chemokine using Milliplex (Millipore, Billerica, Massachusetts, USA). The assays were run according to the manufacturer's guidelines with the exception that the standard and test samples were diluted in the Tissue Homogenate Lysis Buffer rather than the kit-supplied assay diluents.
3
Exosome Purification from FBS
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Differential ultracentrifugation, size-exclusion chromatography, and ultrafiltration have been used in consecutive three steps for exosome purification from FBS, as described [12 (link)]. Differential ultracentrifugation: 20 min centrifugation of FBS (total volume 30 mL) at 2000× g, then 18 h centrifugation of the supernatant at 126,000× g (Type 45 Ti rotor, Beckman Coulter, Pasadena, CA, USA), washing the pellet with PBS and resuspending with PBS, re-pelleting it by 70 min ultracentrifugation at 126,000× g, finally resuspending in 200 μL PBS. Size-exclusion chromatography: 1.1 mL Sephacryl S-1000 gel filtration column (diameter 4.7 mm, height 70 mm). Ultrafiltration: 100-nm pore size ultrafiltration (final exosome purification) using Millipore Ultrafree-MC Centrifugal Filter Device (centrifugation at 12,000× g, 1 min). The size distribution and homogeneity of exosomes have been determined by using Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK), the diameter distribution was 50–110 nm [12 (link)]. Exosomes from neuroblastoma IMR-32 cell culture supernatants were purified as described [12 (link)].
4
Murine Plasma Exosome Isolation
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Murine whole blood samples from tumor-bearing mice were collected with 4 mM EDTA, pH 8.0 (Invitrogen Inc., Carlsbad, CA, USA), and subjected to plasma separation by centrifugation at 3500 × g for 5 min at room temperature. Plasma exosomes were isolated using the ExoQuick exosome precipitation solution (SBI, System Biosciences, CA, USA) according to the manufacturer’s protocol. The extracted exosomes were resuspended in 1 × PBS and sterilized by Ultrafree-MC Centrifugal Filter Devices (0.22 μm, Millipore Co.), and stored at – 80°C for subsequent experiments.
5
Characterization of n-Alkyl-β-D-Maltopyranosides
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The n-alkyl-β-d -maltopyranosides used in these experiments were the following: n-hexyl-β-d -maltopyranoside (catalogue No. A6820,0001, AppliChem, Darmstadt, Germany), n-octyl-β-d -maltopyranoside (catalogue No. A6809,0001, AppliChem), n-nonyl-β-d -maltopyranoside (catalogue No. 59965-1G, Sigma–Aldrich, Hamburg, Germany), n-decyl-β-d -maltopyranoside (catalogue No. D7658, Sigma–Aldrich), n-undecyl-β-d -maltopyranoside (catalogue No. 94206, Sigma–Aldrich), n-dodecyl-β-d -maltopyranoside (catalogue No. D4641, Sigma–Aldrich), n-tridecyl-β-d -maltopyranoside (catalogue No. 16321, Sigma–Aldrich) and n-tetradecyl-β-d -maltopyranoside (catalogue No. A4810,0250, AppliChem).
All detergents were obtained commercially. To prepare sample solutions, solid n-alkyl-β-d -maltopyranosides were dissolved in pure water exceeding the CMC by three to ten times (Table 1 ▶ ) at an ambient temperature of 293 K. Prior to DLS analysis all samples were centrifuged at 16 100g for 60 min (Centrifuge 5415 R, Eppendorf, Hamburg, Germany) and filtered through a 0.2 µm filter (Ultrafree-MC Centrifugal filter devices, 0.5 ml; catalogue No. PR02905, Millipore, Schwalbach, Germany). DLS measurements were performed in replicates of 25 (30 s data-recording time) for each sample investigated and standard errors were estimated from the scatter of the replicates.
All detergents were obtained commercially. To prepare sample solutions, solid n-alkyl-β-
6
Optimized Metabolite Extraction Protocol
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LC–MS-grade methanol (MeOH), acetonitrile (ACN), and methyl tert-butyl ether (MTBE) were purchased from VWR International (Zaventem, Belgium). Ammonium formate (99%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Ultrafree®-MC centrifugal filter devices (0.22 μm) were obtained from Millipore (Bredford, MA, USA). DPPH and ABTS were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Other chemicals and reagents were of analytical grade.
7
Heat-Induced Changes in MRJP1 Oligomer
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Lyophilized MRJP 1 oligomer was gently dissolved in phosphate buffer saline and sterilized using Ultrafree-MC Centrifugal Filter Devices (Millipore). Two hundred microliters of 5 μg/μl MRJP1 oligomer solution was divided into 1.5-ml polypropylene sample tubes (Product No. A151; ASSIST, Tokyo, Japan). Heating was carried out for 30 min using CHILL HEAT CHT-1000 (Asahi Glass Co., Ltd., Tokyo, Japan). Heating temperatures were 56°C, 65°C or 96°C, which are able to denature most non-covalent protein interactions, pasteurize liquid foods and disrupt protein structures, respectively. After heat treatment, samples were immediately subjected to electrophoretic analysis, Superose 12 HPLC analysis, and cell proliferation assay.
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