10−30% glycerol gradients were prepared by mixing 10% glycerol buffer (10% glycerol, 20 mM Tris/HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.3% digitonin) and 30% glycerol buffer (30% glycerol, 20 mM Tris/HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.3% digitonin) using pre-programmed conditions for 10−30% glycerol gradients on Gradient Master (BioComp Instruments). They were subsequently cooled at 4 °C for 2−3 h. Solubilized mitochondria, after being incubated with buffer or Jac1sfGFP for 30 min, were overlayed on the gradients, after which ultracentrifugation was carried out in Sw60Ti rotors (Beckmann Coulter) for 18 h at 121,262 × g at 4 °C. Fractions were collected from the top and were analyzed by SDS-PAGE followed by immunodecoration. Signals were quantified using ImageQuant TL.
Sw60ti rotor
Manufactured by Beckman Coulter
Sourced in United States
The SW60Ti rotor is a swinging-bucket rotor designed for use in high-speed ultracentrifugation. It is compatible with Beckman Coulter's ultracentrifuge systems and is capable of achieving high-g force centrifugation. The rotor's core function is to provide efficient and reliable separation of samples in a controlled environment during the ultracentrifugation process.
Automatically generated - may contain errors
Lab products found in correlation
Sw28 rotor, by Beckman Coulter (7 mentions)
Optiprep, by Merck Group (5 mentions)
Sw55ti rotor, by Beckman Coulter (3 mentions)
Total exosome rna protein isolation kit, by Thermo Fisher Scientific (3 mentions)
Piston gradient fractionator, by BioComp Instruments (3 mentions)
Gradient master, by BioComp Instruments (2 mentions)
Ultracentrifuge tube, by Beckman Coulter (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Optiprep, by Beckman Coulter (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Gradient fractionator, by BioComp Instruments (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Optima l 90k, by Beckman Coulter (2 mentions)
Ferritin, by GE Healthcare (2 mentions)
Aldolase, by GE Healthcare (2 mentions)
Conalbumin, by GE Healthcare (2 mentions)
Sw32ti rotor, by Beckman Coulter (2 mentions)
Gradient master device, by BioComp Instruments (2 mentions)
Polyallomer tube, by Beckman Coulter (2 mentions)
89 protocols using sw60ti rotor
1
Mitochondrial Protein Separation by Glycerol Gradient
2
Isolation of Exosomes from Cell-free Serum
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Cell-free serum with a volume of 500 μL was thawed on ice, overlaid onto a 15% sucrose cushion and ultracentrifuged at 150,000 g for 16 h at 4°C using SW60Ti rotors (Beckman Coulter). Pelleted exosomes were resuspended in 100 μL 1×PBS by overnight incubation at 4°C and then were ultracentrifuged at 150,000 g at 4°C for 2 h. Pelleted exosomes were resuspended in 150 μL of nuclease-free water.
3
Sucrose Gradient Fractionation of Human Serum Proteins
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Example 4
14 sucrose solutions with concentrations from 22.8% to 60% were prepared and layered sequentially in an ultracentrifuge tube (Beckman Coulter Inc., CA) starting with the most concentrated solution. 500 μL of the human serum was loaded on top before ultracentrifugation for 16.5 h at 200 000 g, 4° C. in a SW60Ti rotor (Beckman Coulter Inc.). After centrifugation, 320 μL of 13 fractions were collected starting from the top of the gradient.
The densities of each fractions were determined by weighing a fixed volume. 20 μL of each fractions was resolved on 4-12% SDS-polyacrylamide gels. The gels were either stained with SilverQuest™ Silver Staining Kit (Invitrogen, Carlsbad, Calif.) or electroblotted onto a nitrocellulose membrane. The membrane was probed with either a 1:50 dilution of mouse anti-human CD9 antibody followed by a 1:1250 dilution of a HRP-conjugated donkey anti-mouse IgG antibody. All antibodies were purchased from Santa Cruz.
The bound antibodies were visualized using HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, Mass.) and exposure to an X-ray film. Fractions 9 to 13 from the sucrose gradient were pooled and dialysed against PBS pH7.4 overnight. The pooled fractions were concentrated to 20 μL
4
Liposomal Drug Characterization Protocol
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LUV dispersions were diluted with PBS65 (same buffer used to prepare the LUVs)
1:100 (v/v) and filtered through polyether sulfone membranes (0.2 µm pore size, VWR International, Radnor, USA) prior to size measurement. For the determination of the electrokinetic potential (ZP) on liposomes surface, LUV dispersions were diluted 1:20 (v/v) with freshly filtrated deionized water (0.2 µm pore size filter) and analysis conducted at room temperature (23-25°C) using the DTS1070 cell (Malvern, Worcestershire, UK). The liposomal size and ZP were measured using a Zetasizer Nano Zen 2600 (25°C, Malvern, Worcestershire, UK) as previously described (Wu et al., 2019) (link). To determine the entrapment efficiency (EE), drug-loaded LUVs were separated from the unentrapped drug by ultracentrifugation (200 000 g, 10°C, 30 min, Beckman model L8-70M with SW 60 Ti rotor, Beckman Instruments, California, USA) (Wu et al., 2019) (link). The drug concentrations in both LUVs and supernatant (representing unentrapped drug concentration) were quantified spectroscopically (Spectra Max 190 Microplate, Spectrophotometer Molecular devices, Sunnyvale, USA) using wavelengths at 273 and 247 nm for caffeine and hydrocortisone, respectively. Two samples for each batch of formulations were measured minimum three times. Experiments were repeated in minimum duplicates (n≥2).
1:100 (v/v) and filtered through polyether sulfone membranes (0.2 µm pore size, VWR International, Radnor, USA) prior to size measurement. For the determination of the electrokinetic potential (ZP) on liposomes surface, LUV dispersions were diluted 1:20 (v/v) with freshly filtrated deionized water (0.2 µm pore size filter) and analysis conducted at room temperature (23-25°C) using the DTS1070 cell (Malvern, Worcestershire, UK). The liposomal size and ZP were measured using a Zetasizer Nano Zen 2600 (25°C, Malvern, Worcestershire, UK) as previously described (Wu et al., 2019) (link). To determine the entrapment efficiency (EE), drug-loaded LUVs were separated from the unentrapped drug by ultracentrifugation (200 000 g, 10°C, 30 min, Beckman model L8-70M with SW 60 Ti rotor, Beckman Instruments, California, USA) (Wu et al., 2019) (link). The drug concentrations in both LUVs and supernatant (representing unentrapped drug concentration) were quantified spectroscopically (Spectra Max 190 Microplate, Spectrophotometer Molecular devices, Sunnyvale, USA) using wavelengths at 273 and 247 nm for caffeine and hydrocortisone, respectively. Two samples for each batch of formulations were measured minimum three times. Experiments were repeated in minimum duplicates (n≥2).
5
SNARE Protein Reconstitution into Liposomes
6
Exosome Isolation via Ultracentrifugation
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Exosome isolation was carried out using ultracentrifugation [33 (link)]. In brief, MSC culture supernatants were subjected to successive centrifugations at 3000 × g (30 min) and 10,000 × g (30 min). Exosomes were then pelleted at 64,000 × g for 110 min using an SW28 rotor (Beckman Coulter, Brea, CA, USA). Exosome pellets were resuspended in 0.32 M sucrose and centrifuged at 100,000 × g for 1 h (SW60Ti rotor, Beckman Coulter). The exosome pellet was then resuspended in phosphate-buffered saline (PBS). Nanosight 2000 analysis and transmission electron microscopy (TEM) were used to identify exosomes. RNA and proteins were extracted from exosomes using a Total Exosome RNA & Protein Isolation Kit (Invitrogen, Carlsbad, CA, USA) for further analysis.
7
Exosome Isolation and Labeling
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Example 4
Some of the exosome samples were further isolated using ultracentrifugation. After the conditioned medium was concentrated, the exosomes were labelled with DiR lipophilic dye in the specified dilution. The samples were placed in the 37° C. incubator for 30 minutes. The samples were resuspended in ˜3.5 mL of Plasmalyte and were loaded into ultracentrifuge tubes (11×60 mm tubes). The samples were weighed before centrifugation to ensure that all weights were equivalent. The samples were spun at 110,000 g (39,300 rpm using Beckman Coulter SW 60Ti Rotor) for 70 minutes. After the samples were finished spinning, the supernatant was removed from each tube and was discarded. The remaining pellet was resuspended by vortexing and sonicating briefly in 1 mL of Plasmalyte. Afterwards, 2.5 mL of Plasmalyte was added and the samples were vortexed and sonicated again. The samples were spun a second time at 110,000 g for another 70 minutes. After the second spin, the remaining supernatant was saved as the free dye vehicle. The samples were resuspended again by vortexing and sonicating briefly in 1 mL of Plasmalyte, followed by a second round of resuspension in 2.5 mL of Plasmalyte. A small sample was used to measure particle count on the Nanosight. Once particle count was calculated, the samples were normalized to ˜2e10 particles/mL
8
Cell Fractionation Using OptiPrep Gradient
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Cell fractionation was done on a OptiPrep TM gradient as previously described (Chakrabarty and Bhattacharyya, 2017) . Briefly, a 3-30% or 3-15.5% OptiPrep TM (Sigma) continuous gradient was prepared in a buffer containing 78 mM KCl, 4 mM MgCl 2 , 10 mM EGTA, 50 mM HEPES pH 7.0. Cells were harvested and washed in ice-cold PBS followed by homogenization using a Dounce homogenizer in a buffer containing 0.25 M sucrose, 78 mM KCl, 4 mM MgCl2, 8.4 mM CaCl 2 , 10 mM EGTA, 50 mM HEPES pH 7.0 alongwith 100 ug/ml Cycloheximide, 0.5 mM DTT, 40 U/ml RNase Inhibitor (Applied Biosystems), 1X Protease inhibitor (Roche). The lysate was cleared by centrifugation at 1,000g for 5 min, twice. The clarified lysate was loaded on top of the OptiPrep TM gradient and ultracentrifuged at 36,000 rpm for 5h using Beckman Coulter SW60Ti rotor at 4 0 C. After ultracentrifugation, ten fractions were collected by aspiration from top of the tubes. The fractions were then processed for RNA or protein analysis.
9
Fractionation and Analysis of Extracellular Vesicles
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First, 164K pellets collected by differential centrifugation from 8–15 × 107 cells were resuspended in 1.1 mL of STE buffer (0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) and transferred to the tube of SW60Ti rotor (Beckman) and mixed 1:1 with 60% (wt/vol) stock solution of iodixanol (Optiprep medium, Sigma). STE buffer diluted iodixanol solutions, 1 mL of 20% iodixanol, and 0.8 mL of 10% iodixanol were layered sequentially on the top and the tube centrifuged at 350,000× g and 4 °C for 1 h (brake off). Eight fractions of 480 μL were collected from the top of the tube. For Western blot and CCA in the downstream analyses, 320 μL and 160 μL (respectively) from each 480 μL fraction was diluted with 2 mL and 240 μL PBS (respectively) and centrifuged at 164,000× g for 30 min in TLA110 and TLA 120.1 rotors (Beckman; respectively). For Western blot and immuno-isolation in the downstream analyses, each fraction was split into two 240 μL portions, diluted with 2 mL PBS for Western blot and 560 μL PBS for immuno-isolation, followed by centrifugation in TLA110 and TLA 120.2 rotors (Beckman), respectively. The pellets were resuspended in PBS for immuno-isolation, radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling) for Western blot, or Quick C-Circle Preparation (QCP) lysis buffer [16 (link)] for CCA, and stored at −20 °C.
10
Exosome Isolation and Characterization
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Exosomes were isolated using ultracentrifugation 32 (link) . In brief, MEPM cells culture supernatants were successively centrifuged at 800 g (10 minutes) and 2000 g (30 minutes) at 4°C. Then, exosomes were pelleted at 27300 rpm for 1 hour with an SW60 rotor (Beckman Coulter, California, USA). Exosome pellets were resuspended in phosphate-buffered saline (PBS) and centrifuged at 27300 rpm for 1 hour (SW60Ti rotor; Beckman Coulter). The exosome pellets were resuspended in PBS. Exosomes were identified using Nanosight 2000 analysis and transmission electron microscopy (TEM). Western blot analysis for exosomal markers, including CD9, CD63, Heat Shock Protein 70 (HSP70), and Tumor Susceptibility Gene 101 (TSG101), were used under standard protocols with antibodies (Table1). RNA from exosomes were extracted with a Total Exosome RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA) for further analysis.
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