Polysome profiling was prepared as previously described, 51 with modifications. Approximately 2 × 10 7 Huh7 and HeLa cells were incubated with 400 μM CHX (MCE) for 10 min and then pelleted. Pellets were washed twice in PBS with 400 μM CHX and immediately lysed in 200 µL cold lysis buffer (100 mM KCl, 10 mM MgCl 2 , 50 mM TrisCl, pH 7.4 and 0.5% NP-40) for 10 min on ice and pipetted to homogenize. The lysates were clarified by centrifugation at 700× g for 5 min at 4 °C to discard the cell nucleus and 12,000× g for 10 min at 4 °C to discard mitochondria and debris. Lysates were then loaded onto 10%-50% sucrose gradients and ultracentrifuged in an SW41 Ti swinging-bucket rotor (331362, Beckman) at 36,000 rpm for 2 h at 4 °C. For RNase A digested polysome profiling, 22 lysates containing 100 μg of total RNA were treated with RNase A (ThermoFisher Scientific) at 5 mg/L for 45 min at room temperature and added 400U of RNase Inhibitor (Beyotime Inc.) to terminated the digest reaction. Digested lysates were then loaded onto 10%-40% sucrose gradients and ultracentrifuged in an SW41 Ti swinging-bucket rotor (331362, Beckman) at 36,000 rpm for 2 h at 4 °C. Samples were fractionated using a Biocomp gradient fractionator for absorbance polysome profiles and separation. Control and SMYD5 KO samples were measured and loaded evenly with equivalent 260 nM OD values.
Sw 41 ti swinging bucket rotor
Manufactured by Beckman Coulter
Sourced in United States
The SW 41 Ti Swinging-Bucket Rotor is a centrifugation device designed for high-speed separation of biological samples. It features a swinging-bucket design that allows for the effective separation of particles or molecules based on their density and size. The rotor is compatible with various centrifuge models and can achieve high relative centrifugal forces, making it a useful tool in scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Serum triglyceride determination kit, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Optiprep, by Merck Group (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Fc 203b fraction collector, by Gilson (2 mentions)
Gradient station ip, by BioComp Instruments (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Beckman l8 70m, by Beckman Coulter (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Optima l 100k, by Beckman Coulter (1 mentions)
Aim 5 serum free medium, by Thermo Fisher Scientific (1 mentions)
Millex gv filter, by Merck Group (1 mentions)
Micro bca protein assay, by Thermo Fisher Scientific (1 mentions)
Centrifuge 5430 r, by Eppendorf (1 mentions)
Sw28 swinging bucket rotor, by Beckman Coulter (1 mentions)
Optima xl 100k, by Beckman Coulter (1 mentions)
Lipidtox deep red, by Thermo Fisher Scientific (1 mentions)
Fv1000, by Olympus (1 mentions)
H 7100fa, by Hitachi (1 mentions)
37 protocols using sw 41 ti swinging bucket rotor
1
Polysome Profiling Analysis of Huh7 and HeLa Cells
2
Isolation and Characterization of Human TGRL
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The protocol for obtaining human TGRL (Protocol No. 223062) was approved by the Human Subjects Review Committee/IRB at the University of California Davis. The participants were written informed consent to participate in this study. The informed consent was also approved by the ethics committees/IRB. Postprandial blood samples were obtained 3.5 h after consumption of a moderately high fat meal, which corresponds to the peak elevation in plasma triglyceride concentrations. TGRL were isolated from human plasma at a density of less than 1.0063 g/mL following an 18 h centrifugation at 40,000 rpm in a SW41 Ti swinging bucket rotor (Beckman Coulter, Sunnyvale, CA) held at 14°C within a Beckman L8-70M (Beckman) ultracentrifuge. The top fraction TGRL was collected and dialyzed in Spectrapor membrane tubing (mol wt cut off 3,500; Spectrum Medical Industries, Los Angeles, CA) at 4°C overnight against a saline solution containing 0.01%EDTA. The TGRL lipolysis mix routinely used in experiments was normalized based on triglyceride concentrations and was diluted to contain 150 mg/dL of triglycerides. Total triglyceride content of samples was determined using the serum triglyceride determination kit (Sigma Aldrich cat # TR0100). The kit converts triglycerides to free fatty acids and glycerol and glycerol is assayed enzymatically.
3
Polysome Fractionation and Analysis
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Polysomes were size-fractionated by a 10 mL sucrose density gradient made by 1 mL of each sucrose solution (bottom to top: 50%–5%). Briefly, cells were incubated in the medium containing 100 μg/mL cycloheximide (Sigma-Aldrich, NSW, Australia) for 10 min at 37 °C and were lysed in polysome lysis buffer containing 1% Triton™ X-100 (Sigma-Aldrich). The supernatant was collected by centrifugation and was added to the top of the gradient in an ultracentrifuge tube. The gradient was centrifuged for 2 h (36,000 rpm at 4 °C) using the SW 41 Ti Swinging-Bucket Rotor (Beckman Coulter Life Sciences, NSW, Australia). A total of twelve fractions were collected from the top to the bottom of the gradient using the BR-188 Density Gradient Fractionator (Brandel, MD, USA). All fractions were stored at −80 °C for RNA isolation and qRT-PCR.
4
Production of HIV-1 Pseudoviruses
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The plasmid encoding the NL4-3.LucAM.R−E− backbone was obtained from the NIH AIDS Reagent Program (Manassas, VA, USA). The V504E mutant in JRFL Env (V513E by HXBC2 standard numbering) was made by site-directed mutagenesis, using the aforementioned JRFL Env plasmid and the forward primer (GTG to GAG) and reverse primer (CAC to CTC), sourced from Integrated DNA Technologies, Inc. (Coralville, IA, USA):
Forward: 5′-GA GAA AAA AGA GCA GA G GGA ATA GG-3′
Reverse: 3′-CT CTT TTT TCT CGT CT C CCT TAT CC-5′
Presence of the mutation in the resulting plasmid was confirmed with sequencing by GENEWIZ (South Plainfield, NJ, USA).
HEK293T cells were seeded at 3 million cells per flask, transiently transfected with 4 µg of JRFL WT Env or JRFL V504E Env plasmid DNA, 8 µg of backbone plasmid DNA, and 48 µL PEI (1 mg/mL solution), per the pseudovirus production protocol used in prior studies [22 (link),23 (link),26 (link),28 (link)]. Supernatants were collected 48 h after transfection and purified by filtration on a 100 kDa concentrator, then by a 6–20% iodixanol gradient spun at 30,000 RPM for 2 h using an Optima L-100K ultracentrifuge with SW41 Ti swinging-bucket rotor (Beckman-Coulter; Indianapolis, IN, USA). The fractions containing 13% to 18.6% iodixanol were pooled, frozen, and quantified for p24 content as the produced pseudovirus.
Forward: 5′-GA GAA AAA AGA GCA G
Reverse: 3′-CT CTT TTT TCT CGT C
Presence of the mutation in the resulting plasmid was confirmed with sequencing by GENEWIZ (South Plainfield, NJ, USA).
HEK293T cells were seeded at 3 million cells per flask, transiently transfected with 4 µg of JRFL WT Env or JRFL V504E Env plasmid DNA, 8 µg of backbone plasmid DNA, and 48 µL PEI (1 mg/mL solution), per the pseudovirus production protocol used in prior studies [22 (link),23 (link),26 (link),28 (link)]. Supernatants were collected 48 h after transfection and purified by filtration on a 100 kDa concentrator, then by a 6–20% iodixanol gradient spun at 30,000 RPM for 2 h using an Optima L-100K ultracentrifuge with SW41 Ti swinging-bucket rotor (Beckman-Coulter; Indianapolis, IN, USA). The fractions containing 13% to 18.6% iodixanol were pooled, frozen, and quantified for p24 content as the produced pseudovirus.
5
Prion Purification via Sarkosyl and PTA
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Brain homogenate was
prepared from RML-infected FVB mice at 20% w/v in Ca2+/Mg2+-free PBS. The homogenate was centrifuged for 5 min at 500g at 4 °C, and the pellet was discarded to remove cell
debris from the sample. The supernatant was then split into two aliquots,
and sarkosyl was added to each aliquot to give a final concentration
of 2% w/v sarkosyl. To one sample, 10% PTA solution, pH 7.2, was added
(2% final w/v), while the other was left as a control without PTA.
The samples were then incubated overnight at 37 °C, and the aliquots
were each pipetted onto a two-step sucrose cushion containing 50%
sucrose (8 mL) and 80% sucrose layers (2 mL). All sucrose solutions
contain 0.5% sarkosyl, 50 mM sodium HEPES, and 1 mM sodium azide.
The gradients were centrifuged at 134 000g (20 °C) for 16 h in a SW41 Ti swinging bucket rotor (Beckman-Coulter).
Fifteen aliquots were taken from each gradient and labeled A–O
from low to high density. For fractions A–F, 1 mL samples were
collected; for fractions G–O, 500 μL collected. The pellets
were resuspended in 1 mL of a 2% sarkosyl solution and labeled as
the pellet fraction. All aliquots and gradient pellet samples were
analyzed by Western blot using the P-HRP antibody and developed using
ECL reagents.
prepared from RML-infected FVB mice at 20% w/v in Ca2+/Mg2+-free PBS. The homogenate was centrifuged for 5 min at 500g at 4 °C, and the pellet was discarded to remove cell
debris from the sample. The supernatant was then split into two aliquots,
and sarkosyl was added to each aliquot to give a final concentration
of 2% w/v sarkosyl. To one sample, 10% PTA solution, pH 7.2, was added
(2% final w/v), while the other was left as a control without PTA.
The samples were then incubated overnight at 37 °C, and the aliquots
were each pipetted onto a two-step sucrose cushion containing 50%
sucrose (8 mL) and 80% sucrose layers (2 mL). All sucrose solutions
contain 0.5% sarkosyl, 50 mM sodium HEPES, and 1 mM sodium azide.
The gradients were centrifuged at 134 000g (20 °C) for 16 h in a SW41 Ti swinging bucket rotor (Beckman-Coulter).
Fifteen aliquots were taken from each gradient and labeled A–O
from low to high density. For fractions A–F, 1 mL samples were
collected; for fractions G–O, 500 μL collected. The pellets
were resuspended in 1 mL of a 2% sarkosyl solution and labeled as
the pellet fraction. All aliquots and gradient pellet samples were
analyzed by Western blot using the P-HRP antibody and developed using
ECL reagents.
6
Cushioned-density Gradient Ultracentrifugation for EV Isolation
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We pooled the collected plasma or conditioned medium from VSMCs from each group and used the cushioned-density gradient ultracentrifugation method as described by Li et al. [43 (link)]. In brief, ~1 mL of plasma from each group was diluted with PBS (phosphate-buffered saline) to a total volume of 8 mL, placed over 2 mL of 60% iodixanol solution (SKU:D1556, Millipore Sigma, Bedford, USA) and centrifuged at 110,000× g (k factor of 124) for 2 h at 4 °C using an SW41Ti swinging-bucket rotor (Beckman Coulter, Danvers, USA). Following this step, we carefully aspirated and discarded the top 7 mL and recovered the bottom 3 mL, which contained the EVs fraction. For the second density gradient ultracentrifugation step, we transferred the 3 mL in a new tube, mixed well, carefully overlaid three layers of 20%, 10%, and 5% iodixanol (3 mL each), and centrifuged at 110,000× g for 18 h at 4 °C. We then collected 12 fractions (1 mL each) from the top to the bottom of each tube and pooled fractions 6–10 that are enriched for EVs for downstream experiments. To remove iodixanol from the recovered EVs, we performed a dialysis step in a filtered (0.22 μm) 1× PBS buffer using a 100 kD Float-A-Lyzer G2 Dialysis device (SKU:G235059, Spectrum Lab, Rancho Dominguez, CA) according to the manufacturer’s protocol.
7
Polysome Profiling Protocol
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Cells were incubated with 100 µg mL−1 cycloheximide (CST) in the medium at 37 °C for 10 min and were lysed in polysome lysis buffer containing 1% Triton X‐100 (CST). The polysome lysate was centrifugated and added to the top of the sucrose solutions of ten different densities (bottom to top: 50–5%) filled in an ultracentrifuge tube. The polysomes were size fractionated by ultracentrifuge at 36 000 rpm for 2 h at 4 °C using the SW 41 Ti Swinging‐Bucket Rotor (Beckman Coulter, IN, USA). A total of twelve fractions was collected from top to bottom of the sucrose gradient using the density gradient fractionator (Brandel, MD, USA). The total RNA of each fraction was isolated and subject to qRT‐PCR analysis.
8
Exosome Isolation and Characterization
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The cells were grown to approximately 80% confluence, washed three times with phosphate-buffered saline (PBS), and cultured in AIM V serum-free medium (Thermo Fisher Scientific) for 2 or 3 days. Exosomes were collected from the medium by centrifugation. In brief, the medium was collected and sequentially centrifuged at 300×g for 10 min, 2000×g for 20 min, and 10,000×g for 30 min, and then filtered through a 0.22 μm Millex-GV filter (Merck Millipore, Burlington, MA, USA) to remove cells, cellular debris, and large EVs. The medium was then centrifuged at 210,000×g for 70 min using a Beckman L-70K ultracentrifuge (Beckman Instruments, Brea, CA, USA) with an SW 41 Ti swinging-bucket rotor (Beckman Instruments) [24 (link)]. The supernatant was discarded, and the pellet was washed twice with PBS. The pellet was resuspended in PBS and stored at −80 °C. Protein content was measured using a micro BCA protein assay (Thermo Fisher Scientific). The exosomes were characterized using tunable resistive pulse sensing with a qNano instrument (Meiwafosis Co., Tokyo, Japan) according to the manufacturer's instructions.
9
Yeast Cell Fractionation and Purification
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The applied protocol is adapted from Kurita et al.34 (link) Briefly, 100 mg of yeast cells was suspended in 2 mL of
ice-cold lysis TNE buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10
mM EDTA, supplemented with a 1× complete protease inhibitor cocktail).
The cells were mechanically disrupted with a BeadBug homogenizer in
the presence of 1 g of 0.5 mm glass beads, as previously described.
Subsequently, the cell lysate was filtered from beads that were washed
three times with 1 mL of NaCl 1 M solution. The cell lysate and the
bead washings were pooled and centrifuged at 4 °C for 10 min
at 4000g (Centrifuge 5430 R, Eppendorf): the resulting
pellet was further washed three times with 1 mL of NaCl 1 M solution.
The washed pellet of nuclei and YCW was suspended in 1 mL of the suspension
buffer (10 mM Tris-HCl pH 7.4, 15.25% sorbitol, 10 mM EDTA, supplemented
with a 1× complete protease inhibitor cocktail), to be transferred
to a 12 mL continuous density gradient of Optiprep (D1556; Sigma-Aldrich,
Saint-Louis, MI) (18–48%). Ultracentrifugation was performed
at 4 °C for 19 h at 155 000g using an SW41Ti
swinging bucket rotor (Beckman Coulter, Brea, CA); 24 fractions of
0.5 mL each were sequentially collected from the gradient top.
ice-cold lysis TNE buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10
mM EDTA, supplemented with a 1× complete protease inhibitor cocktail).
The cells were mechanically disrupted with a BeadBug homogenizer in
the presence of 1 g of 0.5 mm glass beads, as previously described.
Subsequently, the cell lysate was filtered from beads that were washed
three times with 1 mL of NaCl 1 M solution. The cell lysate and the
bead washings were pooled and centrifuged at 4 °C for 10 min
at 4000g (Centrifuge 5430 R, Eppendorf): the resulting
pellet was further washed three times with 1 mL of NaCl 1 M solution.
The washed pellet of nuclei and YCW was suspended in 1 mL of the suspension
buffer (10 mM Tris-HCl pH 7.4, 15.25% sorbitol, 10 mM EDTA, supplemented
with a 1× complete protease inhibitor cocktail), to be transferred
to a 12 mL continuous density gradient of Optiprep (D1556; Sigma-Aldrich,
Saint-Louis, MI) (18–48%). Ultracentrifugation was performed
at 4 °C for 19 h at 155 000g using an SW41Ti
swinging bucket rotor (Beckman Coulter, Brea, CA); 24 fractions of
0.5 mL each were sequentially collected from the gradient top.
10
Polysome Profiling of DUX4-Expressing Cells
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Polysome profiling was performed as previously described63 (link),64 (link) with the following modifications. Four 70% confluent 15 cm dishes of MB135-iDUX4 cells per condition were treated with 100 μg/mL cycloheximide for 10 min, transferred to wet ice, washed with ice-cold PBS containing 100 μg/mL cycloheximide, and then lysed in 400 μL Lysis Buffer (20 mM HEPES pH 7.4, 15 mM MgCl2, 200 mM NaCl, 1% Triton X-100, 100 μg/mL cycloheximide, 2 mM DTT, and 100 U/mL SUPERaseIn RNase Inhibitor) per 15 cm dish. The cells and buffer were scraped off the dish and centrifuged at 13,000 rpm for 10 min at 4°C. Lysates were fractionated on a 10%–60% sucrose gradient using the SW 41 Ti Swinging-Bucket Rotor (Beckman Coulter) at 36,000 rpm for 3 h and 10 min. Twenty-four fractions were collected using a Gradient Station ip (BioComp) and an FC 203B Fraction Collector (Gilson) with continuous monitoring of absorbance at 254 nm. RNA from each fraction was extracted using TRIzol LS Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. RT-qPCR was carried out as described above.
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