DNA was mixed with cesium chloride (CsCl), and the mixture was adjusted to a volume of 4.5 ml, and a the density of 1.753 g/ml (i.e. corresponding to a refractive index of 1.404). The DNA-CsCl solution was transferred to Beckman ultracentrifuge tubes, and samples were centrifuged at 30,000 rpm at 22°C for more than 48 h in a SW55 rotor. After centrifugation, the tube was inserted into a gradient collector, a hole was punctured at the bottom of the tube, and fractions of 5 drops each were collected in Eppendorf tubes (up to 50 fractions). The DNA concentration of each fraction was measured using a spectrophotometer, and the refractive index measured using a refractometer, after which the fractions were slot blotted onto a positively charged nylon membrane. The wells of the slot blotter were washed with denaturation buffer (0.5 M NaOH, 0.5 M NaCl). The membrane was then air dried and UV cross-linked. The HPV genomes were detected using DIG-labelled probes (see Southern blot analyses above).
Beckman ultracentrifuge tube
Manufactured by Beckman Coulter
Sourced in United States
Beckman ultracentrifuge tubes are designed for use with ultracentrifugation systems. These tubes are engineered to withstand the high centrifugal forces generated during ultracentrifugation, which is a technique used to separate and analyze various biomolecules and particles based on their size, shape, and density.
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Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Sw 41 ti swing bucket rotor, by Beckman Coulter (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Protease inhibitor cocktail, by Promega (1 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
Rnasin, by Promega (1 mentions)
Optiprep gradient, by Merck Group (1 mentions)
Sw55ti rotor, by Beckman Coulter (1 mentions)
Optima l 100 xp, by Beckman Coulter (1 mentions)
Nanosight ns300 instrument, by Malvern Panalytical (1 mentions)
0.22 μm pore polyvinylidene fluoride filter, by Merck Group (1 mentions)
Nta 3.0 analytical software, by Malvern Panalytical (1 mentions)
Coulter ultracentrifuge, by Beckman Coulter (1 mentions)
Optiprep, by Merck Group (1 mentions)
Sypro orange, by Thermo Fisher Scientific (1 mentions)
11 protocols using beckman ultracentrifuge tube
1
Density Gradient Centrifugation for DNA Separation
2
Subcellular Fractionation of Liver Tissue
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100 mg of liver tissue were lysed in 2.5 ml of lysis buffer A (10 mM KCl, 10 mM Tris–HCl pH 8.0, 2 mM MgCl2 and 0.05% NP-40) with a protease inhibitor cocktail (Roche #04693116001). Following incubation on ice for 20 min, tissues were lysed using a tight-fitting douncer with 40 strokes, followed by centrifugation at 13 000 rpm spin at 4°C for 10 min to remove cell debris. A sucrose gradient was created by adding 2 ml of 36, 29, 22 and 15% sucrose to Beckman ultracentrifuge tubes and then 2 ml of the supernatant sample were added to the sucrose gradient to make up the final volume to 10 ml. A discontinuous gradient of 15–36% was formed and the tubes were centrifuged at 40 000 rpm for 5 h at 4°C (Beckman SW 41 Ti swing-bucket rotor). From the top of each gradient, 800 μl gradient fractions were collected to yield a total of 12 fractions. The Alexa fluorescence intensity of the fractions was measured using TECAN infinite M1000 Pro (TECAN).
3
Acetylation Analysis in QBI-293 Cells
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QBI-293 cells (ATCC, Manassas, VA,
USA) were maintained in Dulbecco’s Modified Eagle’s
Medium (Mediatech Inc., Manassas, VA, USA) containing 10% fetal bovine
serum (FBS) (Atlanta Biologicals, Lawrenceville, GA, USA), 2 mMl -glutamine (Mediatech), 50 units/mL penicillin, and 50 μg/mL
streptomycin (1% penicillin/streptomycin; Thermo Fisher Scientific,
Waltham, MA, USA). Cells were maintained at 37 °C in a humidified
atmosphere (5% CO2) for all experiments. For compound testing,
cells were dissociated with trypsin/EDTA (Thermo Fisher Scientific)
and plated at 6 × 105 cells/well in 6-well plates.
The medium was aspirated after overnight incubation and fresh medium
containing vehicle or test compound was added. After incubating for
4 h, cells were washed once with 1× phosphate-buffered saline
(PBS), pH 7.4 and then lysed in 200 μL RIPA buffer containing
protease inhibitor cocktail, 1 mM PMSF, and 1 μM TSA. Lysed
cells were scraped into 1.5 mL Beckman ultracentrifuge tubes (Beckman,
Brea, CA, USA) and centrifuged at 100000g for 30
min at 4 °C. Following centrifugation, the supernatant from each
sample was collected and analyzed for protein content by BCA assay.
The samples subsequently underwent analysis for acetyl-tubulin and
α-tubulin levels by ELISA, as previously described.39 (link)
USA) were maintained in Dulbecco’s Modified Eagle’s
Medium (Mediatech Inc., Manassas, VA, USA) containing 10% fetal bovine
serum (FBS) (Atlanta Biologicals, Lawrenceville, GA, USA), 2 mM
streptomycin (1% penicillin/streptomycin; Thermo Fisher Scientific,
Waltham, MA, USA). Cells were maintained at 37 °C in a humidified
atmosphere (5% CO2) for all experiments. For compound testing,
cells were dissociated with trypsin/EDTA (Thermo Fisher Scientific)
and plated at 6 × 105 cells/well in 6-well plates.
The medium was aspirated after overnight incubation and fresh medium
containing vehicle or test compound was added. After incubating for
4 h, cells were washed once with 1× phosphate-buffered saline
(PBS), pH 7.4 and then lysed in 200 μL RIPA buffer containing
protease inhibitor cocktail, 1 mM PMSF, and 1 μM TSA. Lysed
cells were scraped into 1.5 mL Beckman ultracentrifuge tubes (Beckman,
Brea, CA, USA) and centrifuged at 100000g for 30
min at 4 °C. Following centrifugation, the supernatant from each
sample was collected and analyzed for protein content by BCA assay.
The samples subsequently underwent analysis for acetyl-tubulin and
α-tubulin levels by ELISA, as previously described.39 (link)
4
Xenopus Interphase Egg Extract Preparation
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Xenopus interphase egg extract was prepared as described [17 (link),43 (link)]. Clarified egg extract (HSS) was prepared from low speed supernatant that was spun in an SW-55 rotor at 55,000 rpm × 45 min. The clarified middle layer was removed and respun for 30 min, and glycerol was added to 5%, aliquoted, and flash-frozen. Xenopus oocyte extracts were prepared from freshly dissected ovaries by disrupting the follicular layer as described above. The defolliculated oocytes were then washed extensively with 1× MMB containing 200 mm sucrose and 1 mm DTT, and the later staged oocytes settled to the bottom (the less dense stage I and II oocytes were mostly lost during the preparation). The oocytes were settled in 13 × 51-mm Beckman ultracentrifuge tubes, and excess buffer was removed. They were then spin-crushed at 35,000 rpm (150,000 × g) for 40 min in an SW-55 rotor. The middle layer was removed with a pipette and respun for 30 min. The middle layer was again removed, glycerol was added to 5% final, and the extract was aliquoted and flash-frozen in liquid nitrogen.
5
Sucrose Gradient Fractionation of Polysome
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Beckman ultracentrifuge tubes were loaded with 2.2 ml of a 10 to 50% sucrose (Sigma-Aldrich) gradient in salt buffer [2 mM tris (pH 7.5), 10 mM NaCl, and 0.5 mM MgCl2] and incubated horizontally at 4°C overnight. Two pieces of mouse testes were homogenized in 1 ml of polysome extraction buffer [20 mM tris (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.5% (v/v) NP-40, protease inhibitor cocktail, RNasin (Promega), and cycloheximide (100 μg/ml)]. After a 10-min centrifugation, clarified lysates were layered onto ultracentrifuge tubes containing 10 to 50% sucrose gradients and centrifuged at 39,000 rpm for 90 min in an SW41 rotor (Beckman Coulter) at 4°C. Gradients were collected in 12 × 1–ml fractions using a Pasteur pipet, and absorbance was measured at 254 nm. RNA was isolated by extraction using TRIzol reagent. RNA quality and amount were determined using an ND-1000 spectrophotometer (Thermo Fisher Scientific).
6
Isolation and Purification of Mouse Hepatic Stellate Cells
7
Preparation of Human Liver Microsomes and Fractions
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Human liver microsomes (HLM) and human liver S9 fractions (HLS9) were prepared using previously published methods [15 (link), 33 (link)]. Briefly, a 200 mg human liver tissue was cut into approximately one by one mm pieces and homogenized in 600 μL PBS buffer (pH 7.4) using a tissue grinder. The sample was centrifuged at 10,000 × g for 30 min, and the supernatant (i.e., HLS9) was collected. To prepare HLM, the supernatant was transferred to Beckman ultracentrifuge tubes and centrifuged at 300,000 × g for 20 min. The pellets were resuspended in PBS using a tissue grinder and collected (i.e., HLM). Protein concentrations were determined using a Pierce BCA protein assay kit. Both HLS9 and HLM samples were stored at −80°C until use. Of note, the same set of liver samples was used for the preparation of both HLM and HLS9 samples; however, different proteomics methods were used for CES1 protein quantifications (i.e., heavy stable isotype internal standard‐based assay for HLM vs. label‐free quantification method for HLS9) [34 (link)].
8
Ultracentrifugation of Serum Extracellular Vesicles
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All ultracentrifugations were performed at 120000 g AVG in Beckman Coulter Optima L-100 XP ultracentrifuge (stopping without break), with centrifugation durations based on a “50 nm cut-off size” adjustment to the centrifugation duration for each rotor as described in Livshits et al. 2015, with additional 5 min added to allow the rotor to come up to speed46 (link). Serum samples were defrosted and transferred to a 5 ml Beckman ultracentrifuge tube (Prod. No. 344057). The serum was then diluted to 4.6 ml with particle-free PBS and 400 µl of 40% iodixanol-PBS was pipetted into the bottom of the tube for Cushion ultracentrifugation (CUC) while the serum was diluted to 5 ml with particle-free PBS for ultracentrifugation (UC). The tubes were centrifuged at 120000 g (RCF avg, 35600 rpm) for 2 hours at 4 °C, using Beckman Coulter SW55ti rotor. For UC, the supernatant was removed and the EV pellet was resuspended in 50–100 µl residual PBS. For CUC the EV sample was resuspended in 5 ml particle-free PBS and centrifuged at 120000 g (RCF avg, 35600 rpm) for 2 hours at 4 °C, using Beckman Coulter SW55ti rotor. The supernatant was removed and the EV pellet was resuspended in 50–100 µl residual PBS.
9
Exosome Isolation and Characterization
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5 mL of serum samples was filtered through a 0.22‐μm pore polyvinylidene fluoride filter (Millipore). Subsequently, the serum was transferred to a fresh Beckman ultracentrifuge tube and PBS was added to make a total volume of 33.8 mL. This was then centrifuged for 120 minutes at 110 000 g, 4°C, using Optima™ XPN ultracentrifuge (Beckman Coulter) to extract the exosomes. Transmission electron microscopy (JEM‐1‐11 microscope, Japan) was used to photograph exosomes at 100 keV, and NanoSight NS300 instrument (Malvern Instruments Ltd. UK) equipped with NTA 3.0 analytical software (Malvern Instruments Ltd. UK) was used to determine the size distribution and concentration of exosomes.
10
Lipid Raft Isolation and Detection
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Cells were treated with 5 ng/mL IL-1β or PBS for 30 min and lysed in cell lysis buffer (CST) containing 1 mM PMSF. An identical amount of protein from each sample was mixed with 90% sucrose in MES buffer (6 mL final volume, sucrose concentration 51.7–58.7%) and transferred to a Beckman ultracentrifuge tube. Four milliliters of 35% sucrose followed by 3 mL of 5% sucrose were overlaid, the samples were spun in a Beckman Coulter ultracentrifuge (39,000 rpm; approximately 180,000 × g in a SW40Ti rotor, 20 h), and 26 fractions were collected from the top of the gradient. For the detection of the lipid raft fractions, all fractions were dot-blotted with HRP-labeled cholera toxin B (Sigma-Aldrich) to detect Ganglioside GM1.
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