The cells grown to confluence in 15-cm dishes were washed with PBS, scraped, pelleted, and centrifuged at ~800 x g for 1 min at 4°C. After discarding the supernatant, SPE Buffer I from the FOCUS™ SubCell kit (G-Biosciences, St Louis, MO, USA) was added to the pellet. The samples were vortexed and incubated on ice for 10 min. Then, the cells were lysed using a Dounce homogenizer with 20 strokes per sample. To pellet the nuclei, the samples were centrifuged at 700 x g for 10 min at 4°C. The supernatant was transferred to a new tube and subsequently centrifuged at 12,000 x g for 15 min at 4°C to pellet the mitochondria. The remaining supernatant was transferred to a new tube and subsequently centrifuged at 100,000 x g for 60 min at 4°C in a SW50.1 swinging bucket rotor (Beckman Coulter™, Palo Alto, CA, USA), to pellet the membrane fraction. The plasma membrane-enriched pellet was subjected to detergent-free sucrose gradient ultracentrifugation (16 (link),23 (link)–26 (link)). Twelve fractions were collected and immunoblotted for N-Myc-tagged D1R and CAV-1.
Sw 50.1 swinging bucket rotor
Manufactured by Beckman Coulter
Sourced in United States
The SW-50.1 swinging bucket rotor is a laboratory centrifugation device designed for high-speed separation of samples. It features a fixed-angle design and can accommodate sample volumes ranging from 1.5 to 50 milliliters. The rotor is compatible with a variety of sample tubes and is suitable for applications requiring efficient separation of cellular and subcellular components.
Automatically generated - may contain errors
4 protocols using sw 50.1 swinging bucket rotor
1
Subcellular Fractionation and Immunoblotting
2
Isolation of Small Extracellular Vesicles by Differential Ultracentrifugation
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We followed the protocol for EV isolation from viscous fluids by differential ultracentrifugation described by Théry et al. [23 (link)] and Caby et al. [24 (link)] with slight modifications. Thawed supernatants were diluted with ice-cold PBS to a final volume of 35 mL and transferred to ultracentrifuge tubes (#326823, Beckman Coulter, Brea, CA, USA) to perform a first ultracentrifugation round at 110,000× g (4 °C) for 3 h using an SW-28 swinging bucket rotor (k factor 245.5; Beckman Coulter). The obtained pellets (containing mostly small EVs) were resuspended in 5 mL of ice-cold PBS (Gibco), transferred to small ultracentrifuge tubes (#326819, Beckman Coulter) and centrifuged again at 110,000× g (4 °C) for 90 min using an SW-50.1 swinging bucket rotor (k factor 154.5; Beckman Coulter). The final cleared pellets were resuspended in 120 μL of ice-cold PBS and aliquoted in Protein LoBind tubes (#0030108434, Eppendorf AG, Hamburg, Germany) for NTA, TEM, protein analysis and RNA extraction. Aliquots were frozen in liquid nitrogen and stored at −80 °C for further analysis.
3
Isolation of Small Extracellular Vesicles
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We used a standard protocol of differential centrifugation described by Thery et al. [14 ] with slight modifications. Briefly, thawed supernatants from UA and UF samples were diluted with ice-cold PBS or saline, respectively, to a final volume of 35 mL and transferred to ultracentrifuge tubes (#326823, Beckman Coulter, Brea, CA, USA) to perform the first ultracentrifugation round at 110,000× g (4 °C) for 2 h with a SW-28 swinging bucket rotor (k factor 245.5; Beckman Coulter). The obtained pellet (containing mostly small EVs) was resuspended in 5 mL of ice-cold PBS and transferred to small ultracentrifuge tubes (#326819, Beckman Coulter) and centrifuged again at 110,000× g (4 °C) for 1 h with a SW-50.1 swinging bucket rotor (k factor 154.5; Beckman Coulter). The final cleared pellet was resuspended in 120 μL of ice-cold PBS (#70011-044, Gibco, Grand Island, NY, USA); aliquoted in Eppendorf tubes (Protein LoBind #022431005) for nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), RNA isolation and protein analysis; frozen in liquid nitrogen and stored at −80 °C for further analysis.
4
Purification of Silver Nanoparticles
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The purification of the raw AgNPs was performed using a Sorvall WX Ultra Series, WX 80 Ultracentrifuge (Thermo Fisher Scientific, MA, USA) with a Beckman SW 50.1 swinging bucket rotor (max. 5 mL) where 3.0 g of the NPs suspension were placed. Samples were vacuum centrifuged using a relative centrifugal field (RCF) of 10,000g, 20,000g, and 30,000g for 20 min with a deceleration time of 5 min at a temperature of 4 °C. Finally, the supernatant from the centrifugation was carefully separated from the pellet deposited at the bottom of the test tubes. Both centrifugation products (supernatant and pellet) were analyzed by TEM, as detailed above.
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