The periplasmic fraction of cultures was extracted following a previously described protocol [37 (link)] from 50 mL of an OD750 nm ≈ 4 culture. The pellet fraction (i.e. spheroplasts) was subjected to further fractionation. Spheroplasts were lysed in lysis buffer without Triton X-100 and broken as described previously (“Preparation of cleared cell lysates ” section). Debris, beads and unbroken cells were collected by centrifugation at 3000×g for 5 min at 4 °C. The subsequent fractionation is based on a previously described protocol [38 (link)]. Briefly, the lysed spheroplasts were centrifuged at 100,000×g, 4 °C for 60 min using a MLS-50 swinging-bucket rotor (Beckman Coulter). The supernatant (containing the cytosolic fraction) was collected in a separate tube and the membrane fraction was resuspended in lysis buffer. An aliquot of the total membrane fraction was kept for further analysis, the rest was loaded on a two-phase sucrose gradient [30% (w/v) and 50% (w/v)] for enrichment of the thylakoid membranes. The sucrose gradient was centrifuged for 60 min at 4 °C and 150,000×g. The thylakoid membranes were collected from the 30 to 50% sucrose interphase.
Mls 50 swinging bucket rotor
Manufactured by Beckman Coulter
Sourced in United States
The MLS-50 Swinging-Bucket Rotor is a laboratory centrifugation equipment designed for the separation and fractionation of biological samples. It features a swinging-bucket design that allows samples to sediment in a horizontal position, providing efficient separation of components. The rotor is compatible with various tube sizes and can achieve high relative centrifugal forces, making it suitable for a range of applications in life science research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Quick seal polypropylene tubes, by Beckman Coulter (2 mentions)
Thinwall polypropylene tube, by Beckman Coulter (2 mentions)
Optima max xp ultracentrifuge, by Beckman Coulter (2 mentions)
0.8 μm filter, by Merck Group (2 mentions)
Optima xe ultracentrifuge, by Beckman Coulter (2 mentions)
Nta 3, by Malvern Panalytical (2 mentions)
Optima max 130k ultracentrifuge, by Beckman Coulter (2 mentions)
Macsquant running buffer, by Miltenyi Biotec (1 mentions)
2k 2k megapixel side mounted tem ccd camera, by Olympus (1 mentions)
Ab15277, by Abcam (1 mentions)
Ab50967, by Abcam (1 mentions)
Ab8592, by Abcam (1 mentions)
Ab62642, by Abcam (1 mentions)
A2066, by Merck Group (1 mentions)
Sc 28534, by Santa Cruz Biotechnology (1 mentions)
Sc 15407, by Santa Cruz Biotechnology (1 mentions)
Ag 20b 0014 c100, by Adipogen (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
11 protocols using mls 50 swinging bucket rotor
1
Subcellular Fractionation Protocol
2
Isolation and Characterization of Extracellular Vesicles from Glioblastoma Cell Line
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Isolation of EV was done from conditioned media after 48 hours of culturing GL261 in RPMI with 1% P/S and 5% EV-depleted FBS (see “Cell Culture ”). The differential ultracentrifugation protocol consisted of subsequent centrifugation at 300×g for 10 min and 2000×g 10 min. Supernatants were filtered through 0.8μm filter (Sigma) and centrifuged for 100,000×g (k-factor of 220.1) 120 min in Quick-Seal® Polypropylene Tubes (Beckman) using Type 70 Ti in Optima XE ultracentrifuge (Beckman) to pellet EVs. To wash and concentrate EVs, pellets were resuspended in remaining supernatant supplemented with OptiMEM and concentrated by centrifugation at 100,000×g (k-factor of 190.7) for 120 min in Thinwall Polypropylene Tubes (Beckman) using MLS-50 Swinging-Bucket Rotor (Beckman) in an Optima Max-XP Ultracentrifuge. Final EV pellet was resuspended in DPBS and characterization of EVs was performed by size distribution analysis using nanoparticle-tracking analysis (NTA 3.2; Malvern), with screen gain set at 3.0 and camera level at 13.0.
Following procedures as described in intracranial tumor implantation method section EVs or an equal volume of carrier fluid (PBS) was injected intracranially. Microglia were isolated 16 and 40 hours after injection of EVs or DPBS following procedures, as previously described.
Following procedures as described in intracranial tumor implantation method section EVs or an equal volume of carrier fluid (PBS) was injected intracranially. Microglia were isolated 16 and 40 hours after injection of EVs or DPBS following procedures, as previously described.
3
Extraction of Gut Microbiota from Fecal Samples
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The study sample comprised 5 fecal samples from healthy donors. No donors reported any severe diseases in the last 6 months. Fresh fecal material was collected in a sterile container and immediately manipulated and homogenized within a maximum time span of 2 h. Nine millilitres of sterile NaCl 0.9% (w/v) was added to 1 g of sample, and the mixture was homogenized in a sterile bag, using a laboratory paddle blender (Stomacher Lab Blender 400, Seward Ltd. UK) for 1 min. Microbiota extraction was then performed following the protocol described by Hevia and coworkers42 (link). A solution of Nycodenz 80% (w/v) (Progen Biotechnik GmbH, Heidelberg, Denmark) was prepared in ultrapure water, and sterilized at 121 °C for 15 min. A volume of 3 mL of the diluted, homogenized fecal sample was placed on top of 1 mL of the Nycodenz solution, and centrifuged for 40 min at 4 °C (10,000×g, MLS-50 Swinging-Bucket Rotor, Beckman Coulter, Indianapolis, IN). The upper phase (soluble debris) was discarded after centrifugation, and the layer corresponding to the microbiota was collected, washed once and resuspended in 1 mL of FC buffer (1 × MACSQuant Running Buffer, MILTENYI BIOTEC, Germany).
4
Isolation and Characterization of Extracellular Vesicles from Glioblastoma Cell Line
5
Isolation of Extracellular Vesicles via Ultracentrifugation
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For the isolation of EVs, cells were grown in 10-cm dishes and conditioned media obtained and cleared by a first centrifugation at 1500 rpm for 5 min at 4°C to eliminate cellular debris. Equal volumes of cleared conditioned media were subjected to ultracentrifugation at 100 000 g (45 000 rpm) in MLS-50 Swinging-Bucket Rotor (Beckman Coulter, cat. no. 367280) mounted on the Optima MAX-130K Ultracentrifuge (Beckman Coulter) for 2 h at 4°C. The floating (S100) and pellet (P100) fractions were then subjected to further analysis.
6
Electron Microscopy of Exosomes
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In experiments with UC, exosomes were re-pelleted in MLS-50 swinging-bucket rotor (Beckman Coulter, Brea, CA, US). Formalin (4%) was layered carefully onto the pellets. After formalin was washed out, pellets were post-fixed with 1% osmium tetroxide (OsO4) for 20–30 min, and block stained with 1% uranyl acetate (in 50% ethanol) for 20–30 min. Then, pellets were dehydrated by graded ethanol for 5 min in 70%, 90%, 96%, and 3×5 min in 100% ethanol. Pelleted samples were embedded in Taab 812 (Taab Laboratories, Aldermaston, UK). Then ultrathin sections were prepared.
Exosome samples obtained by SEC were fixed by buffered 1% OsO4 solution in 1:1 for 30 min. Formvar-coated TEM grids were placed on top of 5–10 μL drops of these fixative-containing samples for 20 min, then grids were consecutively transferred to drops of distillated water (3x5 min), 1% uranyl-acetate in 50% alcohol for contrast staining for 15 min and finally to drops of distilled water for 3x5 min. Grids carefully removed from the last drop of water were air dried and analyzed.
Prepared samples were analyzed under Hitachi 7100 electron microscope equipped by Veleta, a 2k×2k MegaPixel side-mounted TEM CCD camera (Olympus, Tokyo, Japan). Contrast and brightness of electron micrographs were edited in Adobe Photoshop CS3 (Adobe Systems Incorporated, San Jose, CA, US).
Exosome samples obtained by SEC were fixed by buffered 1% OsO4 solution in 1:1 for 30 min. Formvar-coated TEM grids were placed on top of 5–10 μL drops of these fixative-containing samples for 20 min, then grids were consecutively transferred to drops of distillated water (3x5 min), 1% uranyl-acetate in 50% alcohol for contrast staining for 15 min and finally to drops of distilled water for 3x5 min. Grids carefully removed from the last drop of water were air dried and analyzed.
Prepared samples were analyzed under Hitachi 7100 electron microscope equipped by Veleta, a 2k×2k MegaPixel side-mounted TEM CCD camera (Olympus, Tokyo, Japan). Contrast and brightness of electron micrographs were edited in Adobe Photoshop CS3 (Adobe Systems Incorporated, San Jose, CA, US).
7
Isolation and Characterization of Dystroglycan Complex
8
Protein Complex Isolation and Analysis
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Equal amounts of muscle homogenates were layered on a linear 10–40% glycerol gradient containing 20 mM Tris, pH 7.6, 5 mM EDTA, pH 7.4, 100 mM KCl, 1 mM DTT, 0.24% sodium deoxycholate, and 1 mM sodium orthovanadate. Following centrifugation at 131,300 × g for 24 h at 4 °C using a MLS-50 Swinging-Bucket rotor (Beckman Coulter, Brea, CA) to sediment protein complexes, 300 μl fractions were collected, and alternate fractions were subjected to trichloroacetic acid precipitation (10%) for 24 h at 4 °C. Precipitates were then analyzed by SDS-PAGE and immunoblotting.
9
NLRP3 Localization in LPS-Activated iBMDMs
10
Analyzing HSPB1 and Huntingtin Oligomerization
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HSPB1 and huntingtin oligomerization status was analysed by isopycnic ultracentrifugation on a discontinuous sucrose gradient (5–20% w/v), as described by Martin and Ames (116 (link)), with some slight modifications. Briefly, cytoplasmic extracts were prepared with reassembly buffer (100 mM Tris, 0.5-mm MgSO4, 1-mm EGTA, 2-mm dithiothreitol; pH 6.8), containing protease inhibitors and phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany). Equal amounts of protein were loaded on a 5–20% sucrose gradient and centrifuged at 100 000 g (45 000 rpm) in MLS-50 Swinging-Bucket Rotor (Beckman Coulter, cat. no. 367280) mounted on the Optima MAX-130K Ultracentrifuge (Beckman Coulter) for 24 h at 4°C. Twelve fractions (450 ml each) were collected from the top to the bottom of the gradient. The remaining pellets were resuspended in sample buffer (50 μl) and subjected to immunoblot analysis as above indicated.
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