The clarified virus culture supernatant of 50 mL was inactivated with 3 mM binary-ethylenimine (Sigma-Aldrich) treatment at 26 °C for 24 h and concentrated by polyethylene glycol (PEG, MW 6000, Sigma-Aldrich) precipitation. The precipitate was resuspended in tris-KCl (TK) buffer (pH 7.6) and then layered onto 15–45% sucrose density gradients (SDGs) and ultracentrifuged at 100,000× g for 4 h at 4 °C using a SW41Ti rotor(Beckman Coulter, Brea, CA, USA). The ultra-centrifuged SDG was fractionated using a continuous density gradient fractionator (Teledyne ISCO, Lincoln, NE, USA) [22 (link)], and the absorbance of each fraction at 254 nm was consecutively recorded with the spectrophotometer component of the instrument. The area under the peak for specific fractions was measured to calculate the quantity of 146S antigens (µg/mL) according to a previous study [22 (link)]. Each experiment was carried out in triplicate.
Mw 6000
Manufactured by Merck Group
Sourced in United States
The MW 6000 is a laboratory equipment used for molecular weight determination. It is a device designed to measure the molecular weight of various substances, such as polymers, proteins, and other macromolecules. The MW 6000 utilizes advanced technology to provide accurate and reliable measurements, allowing researchers and scientists to better understand the properties and characteristics of the samples being analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Binary ethylenimine, by Merck Group (1 mentions)
Continuous density gradient fractionator, by Teledyne (1 mentions)
Sw41ti rotor, by Beckman Coulter (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Curcumin powder, by Alpha Chemika (1 mentions)
Mw 40000, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
8 protocols using mw 6000
1
Purification and Quantification of 146S Antigens
2
Exosome Purification from Cell Culture Supernatant
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After cells were cultured with exosome-depleted serum (Shin Chin Industrial, SCI), the exosomes were purified from the conditioned medium according to the instructions [16 (link)]. The medium was centrifuged at 500 g for five minutes and at 2000 g for thirty minutes at 4 °C to remove cellular debris and large apoptotic bodies. After centrifugation, media was added to an equal volume of a 2× polyethylene glycol (PEG, MW 6000, Sigma, 81,260) solution (final concentration, 8%). The samples were mixed thoroughly by inversion and incubated at 4 °C overnight. Before the tubes were tapped occasionally and drained for five minutes to remove excess PEG, the samples were further centrifuged at maximum speed (15,000 rpm) for 1 h at 4 °C. The resulting pellets were further purified using 5% PEG and then stored in 50–100 μl of particle-free PBS (pH 7.4) at − 80 °C. The average yield was approximately 300 μg of exosomal protein from 5 ml of supernatant. Total RNA was extracted by using Trizol reagent (Life Technologies), followed by miRNA assessment by microarrays and RT-PCR described below. Exosomes were analysed by electron microscopy to verify their presence, by a nanoparticle characterization system to measure their size and concentration, and by western blot to detect their proteins (HSP70, TSG101, CD63 and CD81).
3
Isolation of HDL from Serum
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Serum stored at −80°C was thawed. HDL was isolated by removing apoB containing lipoproteins from serum using polyethylene glycol (PEG, MW 6000, Sigma Aldrich; St. Louis, MO). 400 μl PEG (20% in water) was added to 1 ml serum, incubated for 20 min at RT, and then centrifuged at 10,000 RPM for 30 min.
4
Isolation of HDL from Serum
5
Serum βhCG Quantification by RIA
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Serum βhCG was quantified by radio-immunoassay. A murine monoclonal antibody specific for βhCG was employed for this purpose. Briefly, diluted sera were incubated with the antibody, 4% normal horse serum and 125I-hCG (40 μCi/μg, ≅ 10,000 cpm) at 4°C for 16 hrs. Polyethylene glycol (Mw 6000; Sigma) at a final concentration of 12.5% was then added. Centrifugation was carried out at 1840 g at 6°C to precipitate immune complexes. Supernatants were decanted and radioactivity in the pellet determined in a gamma counter. Pure hCG (1.25 ng/ml – 40 ng/ml) was employed as standard and serum βhCG was quantified by comparisons with the standard curve obtained upon linear regression.
6
Exosome Isolation and Purification Protocol
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The cell medium was centrifuged at 500 g for five minutes and at 2000 g for thirty minutes at 4 °C to remove cellular debris and large apoptotic bodies. After centrifugation, media was added to an equal volume of a 2× polyethylene glycol (PEG, MW 6000, Sigma, 81,260) solution (final concentration, 8%). The samples were mixed thoroughly by inversion and incubated at 4°C overnight. Before the tubes were tapped occasionally and drained for five minutes to remove excess PEG, the samples were further centrifuged at maximum speed (15,000 rpm) for 1 h at 4°C. The resulting pellets were further purified using 5% PEG and then stored in 50–100 μl of particle-free PBS (pH 7.4) at − 80°C. The average yield was approximately 300 μg of exosomal protein from 5 ml of supernatant. Exosome was re-suspended in 150ul RIPA buffer (Beyotime, P0013B) and incubated on ice for 1h. The lysate was centrifuged for 20min at 16000g at 4°C, and the supernatant was further concentrated by ultracentrifuge filter (Millipore, 3kDa cutoff). Protein concentration was measured by BCA assay (Beyotime, P0012).
7
Curcumin Nanoparticle Synthesis Protocol
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Curcumin powder was purchased from Alpha Chemika (Mumbai, India). The coating agents; polyvinylpyrrolidone (PVP; Mw 40,000) and polyethylene glycol (PEG; Mw 6000), as well ascell staining chemicals; Hoechst (H6024-10ML) and fluorescein isothiocyanate (FITC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was obtained from Fisher Scientific (Loughborough, UK). Acetone and all other reagents were of analytical grade and used as received. The used deionized water (DIH 2 O) used to was ultra-purified from Millipore Milli-Q system (resistivity ~80 MΩ cm).
8
Exosome Isolation and Characterization
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After cells were cultured with exosome-depleted serum (AusGeneX), the exosomes were puri ed from the conditioned medium according to the instructions [23] . The medium was centrifuged at 500 g for 5min and at 2000 g for 30min at 4 °C to remove cellular debris and large apoptotic bodies. After centrifugation, media was added to an equal volume of a 2× polyethylene glycol (PEG, MW 6000, Sigma, 81260) solution ( nal concentration, 8%). The samples were mixed thoroughly by inversion and incubated at 4 °C overnight. Before the tubes were tapped occasionally and drained for 5min to remove excess PEG, the samples were further centrifuged at maximum speed (15,000 rpm) for 1 h at 4 °C. The resulting pellets were further puri ed using 5% PEG and then stored in 50-100 μl of particle-free PBS (pH 7.4) at -80 °C.
The average yield was approximately 300 μg of exosomal protein from 5 ml of supernatant. Total RNA was extracted by using Trizol reagent (Life Technologies), followed by miRNA assessment by microarrays and RT-PCR described below. Exosomes were analyzed by electron microscopy to verify their presence, by a nanoparticle characterization system to measure their size and concentration, and by western blot to detect their proteins (TSG101, CD63 and CD81).
The average yield was approximately 300 μg of exosomal protein from 5 ml of supernatant. Total RNA was extracted by using Trizol reagent (Life Technologies), followed by miRNA assessment by microarrays and RT-PCR described below. Exosomes were analyzed by electron microscopy to verify their presence, by a nanoparticle characterization system to measure their size and concentration, and by western blot to detect their proteins (TSG101, CD63 and CD81).
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