20 × 106 exponentially growing Jurkat cells were electroporated as for luciferase reporter assays. After 24 h, cells were either left nonstimulated or stimulated with anti-CD3 for 2 min. Cells were then centrifuged; resuspended in 1 mL of hypotonic buffer [10 mM Tris–HCl (pH 7.5), 38 mM sodium chloride, 1 mM sodium orthovanadate (Cat. #S6508; Sigma), 1 mM sodium fluoride (Cat. #S7920; Sigma), and CØmplete Protease Inhibitor Cocktail (Cat. #11836145001; Roche, Penzberg, Germany)]; and kept on ice for 5 min. After five freeze/thaw cycles (4 min at −80 °C and 20 s at 42 °C), cells were centrifuged at 14,000 rpm for 10 min at 4 °C and the pellets were discarded. The supernatants were then centrifuged in an ultracentrifuge (Optima TL Ultracentrifuge; Beckman Coulter, Brea, CA, USA) at 60,000 rpm for 1 h at 4 °C using polycarbonate centrifuge tubes (Cat. #349622; Beckman Coulter). The supernatant was considered as the cytoplasmic compartment and Triton X-100 (Cat. #X100; Sigma) was added to a final 1% concentration. The pellet was resuspended again in hypotonic buffer and centrifuged as above. This new pellet was resuspended in hypotonic buffer with 1% Triton X-100 to obtain the plasma membrane fraction. SDS-PAGE buffer was added to all the samples, boiled for 5 min, and analyzed by immunoblot as indicated above.
Polycarbonate centrifuge tube
Manufactured by Beckman Coulter
Sourced in United States
Polycarbonate centrifuge tubes are durable, reusable containers designed for use in centrifugation processes. They are made of polycarbonate, a robust and transparent material that can withstand high-speed centrifugation. These tubes are suitable for a variety of laboratory applications that require sample separation and concentration.
Automatically generated - may contain errors
Lab products found in correlation
Tl 100 ultracentrifuge, by Beckman Coulter (2 mentions)
Optima tl ultracentrifuge, by Beckman Coulter (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Sodium fluoride, by Merck Group (1 mentions)
Prism 9, by GraphPad (1 mentions)
Sw32ti rotor, by Beckman Coulter (1 mentions)
Amicon stirred cell, by Merck Group (1 mentions)
Sw41ti, by Beckman Coulter (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Beckman sw41 rotor, by Beckman Coulter (1 mentions)
Brij o10, by Merck Group (1 mentions)
Gradient master, by BioComp Instruments (1 mentions)
Histodenz, by Merck Group (1 mentions)
Optima tlx, by Beckman Coulter (1 mentions)
Optiprep, by Axis-Shield (1 mentions)
14 protocols using polycarbonate centrifuge tube
1
Protein Extraction from Jurkat Cells
2
Quantification of Pelletable Tau
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Multiple replicas of the reactions were also carried out for the quantification of pelletable tau. At 0, 60, 90, 120, 360 and 720 min, the entire volume of individual reaction replicas was collected for ultracentrifugation. Reactions were centrifuged at 400,000g at 20 °C for 15 min in polycarbonate centrifuge tubes (Beckman Coulter). The pellets were resuspended in 40 μl reaction buffer, to match the volume of the supernatants. Loading buffer was added to supernatants and pellets, which were then heated for 5 min at 95 °C, and 1.5 μl of each was run by SDS–PAGE (4–20% Tris-glycine gels). Band intensities were quantified using ImageJ and data were plotted using Prism 9.5.1 (GraphPad Software).
3
Polysome Profiling of Arabidopsis Transcripts
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Polysome profiling assays were performed essentially as described previously (Missra and von Arnim 2014 (link)), with some modifications. Briefly, total proteins were extracted from 9-days-old WT, trm21-1, trm21-2, TRM21pro::TRM21/trm21-1 and TRM21-3/20 seedlings. Then, 800 µL of supernatant was loaded onto a 36 mL continuous sucrose gradient in a polycarbonate tube and spun in a Beckman SW 32 Ti rotor at 17,000 g at 4 °C for 5 h. Sucrose gradients (15–60%) were prepared by layering 15% (w/v) sucrose on top of an equal volume of 60% (w/v) sucrose in 38 mL polycarbonate centrifuge tubes (Beckman Coulter). Eleven fractions were collected by carefully pipetting samples from the top of the gradient. The polysomal and non-polysomal fractions were determined based on UV absorption profiles obtained from identical but separate experiments. The top seven fractions (1–7) contained ribosome-free mRNAs and monosomes, and the bottom four fractions (8–11) contained mRNAs associated with multiple ribosomes. The RNA from the bottom four fractions was extracted using RNAZol reagent (MRC) and reverse transcribed using random hexamers. RT‑qPCR was performed as above, with ACTIN transcripts used as a negative control. The primers are listed in Suppl. Table S1 .
4
Ultracentrifugation of Concentrated Retentate
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The designated UF filtered supernatant portion was concentrated 50-fold in 50 mM HEPES buffer in a 400 ml capacity Amicon® stirred cell (MilliporeSigma, United States) ultradiafiltration system using a 500 kiloDalton (kDa) molecular weight cut off (MWCO) polyethersulfone (PES) ultrafiltration disk (MilliporeSigma, United States). The concentrated retentate was collected and divided into polycarbonate centrifuge tubes (Beckman) before ultracentrifugation at 40,000 RPM for 2 h at 4°C. The pellet was resuspended in 50 mM HEPES buffer and stored at −20°C until further use (Yaron et al., 2000 (link)).
5
Extracellular Vesicle Isolation from Cell Culture
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HEK293T Cells were plated in a 10 cm or 15 cm tissue culture plate at 50% confluency with either 10 mL or 25 mL of media, respectively, for 48 h before media was collected. THP-1 cells were cultured in flasks at a concentration range of 400,000 to 600,000 cells per mL. EVs were isolated in a 15 mL or 50 mL conical which was centrifuged in a tabletop centrifuge at 2000× g for 20 min at 4°C. The supernatant was collected and added to either Beckman Coulter polycarbonate centrifuge tubes (#349622) or (#344058) and spun at 10,000× g with either SW41 TI or SW28 Beckman rotors, respectively, in an Optima L-90K ultracentrifuge at 4°C for 30 min. Subsequently, the supernatant was collected and ultracentrifuged at 100,000× g for 150 min at 4°C using new centrifuge tubes, as above. Afterwards, the supernatant was discarded, and the pellet was resuspended in PBS. The resuspended pellet was subjected to another round of 100,000× g centrifugation with same rotor and machine for 150 min at 4°C. The supernatant was discarded, and the pellet was resuspended overnight in 100 µL of PBS on an orbital shaker. The resuspended pellets were stored at 4°C and used within 2 weeks. Resuspended EVs were visually inspected before use to verify that a new pellet had not formed before use.
6
Protein Binding of EF-24 in Biological Fluids
7
Preparation of Amyloid Beta Peptide
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Human amyloid beta (Aβ) 42 peptide was purchased from Peptide Institute Inc (Japan), already treated with trifluoroacetic acid and lyophilized powder form before use as described previously [35 (link), 54 (link), 55 (link)]. We kept Aβ 42 peptide at r.t. for 30 minutes and then dissolved using filtered 0.1% NH3 solution (Sigma-Aldrich, USA) according to manufacturer`s instruction. The preparation was ultra-centrifuged using TL-100 ultracentrifuge incorporating TLS 55 rotator (Beckman, USA) at 4°C, 55000 rpm for 3 hr. After ultra-centrifugation, supernatant was collected without touching bottom of polycarbonate centrifuge tubes (Beckman, USA), aliquoted into micro-tubes and quantified by Bradford protein assay method [62 (link)]. Aliquoted tubes were immediately dipped into liquid N2 and finally stored at—80°C as stock solution up to one month. During experiments frozen samples were thawed and diluted with buffer solution (PBS, pH 7.4). Thawed samples were consumed in one experiment and repetitions of freeze-thaw cycles were strictly avoided.
8
Isolation of Extracellular Vesicles from Conditioned Media
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Using 70 mL polycarbonate centrifuge tubes (Beckman Coulter, Brea, CA, United States; cat#:355655), conditioned media was spun using a Ti45 fixed-angle rotor in an Optima XE-90 ultracentrifuge at ~118,000 g/39,000 rpm for 1 h 34 min with max acceleration and deceleration at 4°C. Additional spins were performed as needed by pouring off supernatant and adding remaining conditioned media until the entire volume was processed into a pellet. The resulting pellet was washed for 30 min at 4°C in 2–3 mL of ice-cold 0.1 μm sterile-filtered 1X PBS on a shaker, followed by another spin and one final wash/spin to remove secreted proteins and other components. After the final spin, the supernatant was discarded, and the EV pellet was resuspended in 3 mL of 1X PBS by incubating at 4°C on a shaker for 45 min-1 h. The resulting EV suspension was then concentrated down to 500 μL using a Amicon Ultra-4 3 kDa centrifugal filter unit (MilliporeSigma, Burlington, MA, United States; cat#:UFC800396) that was previously primed with 3 mL of PBS that was centrifuged out to remove particulates from the filter.
9
Lipid Raft Isolation by Flotation Assay
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To isolate lipid rafts, a flotation assay was performed as described previously. 26 Briefly, 10% homogenates were prepared from uninfected mouse brains using citrate buffer (20 mmol/L sodium citrate and 137 mmol/L NaCl, pH 6.0). The homogenates were centrifuged at 1000 Â g for 5 minutes at 4 C to remove cell debris and nuclei. The supernatants were mixed with 1Â volume of ice-cold Brij O-10 extraction buffer [0.1% Brij O-10 (Sigma-Aldrich), 20 mmol/L sodium citrate, and 137 mmol/L NaCl, pH 6.0) and incubated on a rocker at 4 C for 30 minutes, and then mixed with 1.4Â volume of 60% Optiprep (Axis-Shield, Dundee, UK). In 12 mL polycarbonate centrifuge tubes (Beckman Coulter, Brea, CA), 9 mL of 0% to 30% Optiprep continuous gradients were prepared by Gradient Master (BioComp Instruments, Fredericton, NB, Canada), according to the manufacturer's instruction, and underlaid with 2 mL of the Brij O-10 extracted samples. The gradients were centrifuged in a prechilled Beckman SW41 rotor (Beckman Coulter) at 40,000 Â g for 130 minutes at 4 C. The samples were split to 18 fractions (600 mL each) from top (fraction 1) to bottom (fraction 18) and boiled with the addition of 150 mL of 5Â Laemmli's sample buffer.
10
TRAPP Complex Membrane Binding Assay
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