L7 65 ultracentrifuge

Manufactured by Beckman Coulter

Sourced in United States

The L7-65 Ultracentrifuge is a high-performance centrifuge designed for advanced applications in research and clinical laboratories. It is capable of achieving high centrifugal forces, enabling the separation and analysis of a wide range of biological samples, including cells, organelles, and macromolecules.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Polycarbonate centrifuge bottles, by Beckman Coulter (1 mentions) Ultimate 3000 nano lc system, by Thermo Fisher Scientific (1 mentions) Microtof q 2 mass spectrometer, by Bruker (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Elisa, by Fujirebio (1 mentions) 0.2 μm membrane filters, by Thermo Fisher Scientific (1 mentions) Bca assay kit, by Thermo Fisher Scientific (1 mentions)

7 protocols using l7 65 ultracentrifuge

Isolation and Characterization of RBC Membranes

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An expired unit of donor packed red blood cells was acquired from the UC
Davis Medical Center hospital transfusion services, and the provision approved
by the Department of Pathology Clinical Research Oversight Committee Internal
Review. RBC membrane was prepared according to previous studies with
modification^{16 , 34 (link)}. Briefly, the RBCs were lysed
in the hypotonic medium (0.25×PBS) for 90 mins on ice. Samples were
centrifuged at 80000 × g for 90 min with a Beckman L7-65 Ultracentrifuge.
The supernatant was removed and the pink pellet was re-suspended in water. The
membrane protein concentrations were quantified using BCA protein assay kit
(Pierce, Rock-ford, IL).

Xu X., Yang G., Xue X., Lu H., Wu H., Huang Y., Jing D., Xiao W., Tian J., Yao W., Pan C.X., Lin T.Y, & Li Y. (2018). A polymer-free, biomimicry drug self-delivery system fabricated via a synergistic combination of bottom-up and top-down approaches. Journal of materials chemistry. B, 6(47), 7842-7853.

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Virus Protein Isolation and Mass Spectrometry

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The supernatant (50 ml) from virus cultured in Vero cells was collected, and cell debris was removed by centrifugation at 1000 ×g for 15 min at 4 °C. The viruses in the supernatant were concentrated by ultracentrifugation in polycarbonate centrifuge bottles (no. 355603, Beckman Coulter, Brea, CA, USA) using a Beckman L7–65 ultracentrifuge (rotor 70.1 Ti) set at 35,000 rpm for 1.5 h at 4 °C. The resulting pellets were mixed with lysis buffer containing 1% NaCl, 1% sodium dodecyl sulfate (SDS), and 1% Triton-X to produce a virus protein lysate, which was then processed for mass spectrometric analysis via LC-MS/MS by a MicroToF Q II mass spectrometer (Bruker, Germany) coupled to an Ultimate 3000 nano-LC system (Dionex, Sunnyvale, CA, USA) as previously described [24 (link)].

Kosoltanapiwat N., Tongshoob J., Ampawong S., Reamtong O., Prasittichai L., Yindee M., Tongthainan D., Tulayakul P, & Boonnak K. (2022). Simian adenoviruses: Molecular and serological survey in monkeys and humans in Thailand. One Health, 15, 100434.

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Aqueous Phase Rheological Properties

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From each of the three individual sponge cake batters, six samples (20.0–30.0 g) in 38 mL thick polycarbonate tubes (Beckman Coulter, Brea, CA, USA) were centrifuged at 165,000 g (25 °C, 65 min) in a Beckman Coulter L7-65 Ultracentrifuge. From top to bottom, three phases were distinguished; a lipid, an aqueous, and a solid phase. The upper lipid phase was gently removed, and the aqueous phase was carefully decanted. The combined aqueous phase of each of the six samples per batter is further referred to as the BL.
The η_BL of each individual batter was determined in duplicate at 25 °C with a Brookfield Engineering (Middleboro, MA, USA) DV-III Ultra rheometer equipped with a cylindrical SC4-18 spindle (diameter 17.48 mm, length 31.72 mm). The cylindrical spindle was fully immersed in a BL aliquot (6.7 ml) in a SC4-13R sample chamber (diameter 19.05 mm) and then rotated at 10 rotations/min for 150 s. From 60 s onwards, the viscosity was recorded every 15 s for 90 s resulting in seven η_BL measurements per individual batter. The 14 readings of the η_BL of each tested batter were averaged.

Godefroidt T., Riley I.M., Ooms N., Bosmans G.M., Brijs K, & Delcour J.A. (2023). Sucrose substitution in cake systems is not a piece of cake. NPJ Science of Food, 7, 52.

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Blood Separation by Density Gradient

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Blood was washed three times in PBS-0.5% (wt/vol) Bovine Serum Albumin (BSA, Sigma Aldrich), 1 mM EDTA (Ethylene Diamine Tetra Acetate, Sigma Aldrich). Red cells were then layered on top of an OptiPrep^TM/PBS Density Gradient (1.083, 1.087, 1.091, 1.095, 1.100 g/mL), and ultracentrifuged at 141,000g for 30 min at RT (Beckman L7–65 Ultracentrifuge).

Blanc L., Papoin J., Debnath G., Vidal M., Amson R., Telerman A., An X, & Mohandas N. (2015). Abnormal erythroid maturation leads to microcytic anemia in the TSAP6/Steap3 null mouse model. American journal of hematology, 90(3), 235-241.

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HIV-1 Virus Isolation and Quantification

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The HIV-1 BaL isolate was propagated onto primary macrophages and collected from the culture supernatants. The HIV-1 IIIB isolate was obtained from H9/HTLV-IIIB supernatants (Popovic et al., 1984 (link)). The viruses were concentrated by ultracentrifugation at 28,000 rpm for 90 min at 4°C (L7-65 ultracentrifuge; Beckman Coulter), and the virus pellet was suspended in RPMI 1640 medium. The levels of p24 antigen were determined by ELISA (InnoGenetics), and virus input into assays was a function of p24 antigen concentration.

Gerber P.P., Cabrini M., Jancic C., Paoletti L., Banchio C., von Bilderling C., Sigaut L., Pietrasanta L.I., Duette G., Freed E.O., de Saint Basile G., Moita C.F., Moita L.F., Amigorena S., Benaroch P., Geffner J, & Ostrowski M. (2015). Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate. The Journal of Cell Biology, 209(3), 435-452.

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Isolation and Characterization of Extracellular Vesicles

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Tumor cell lines were seeded at 2–2.5 × 10⁶/mL in RPMI/0.5% (vol/vol) BSA and cultured for 48 h at 37 °C, 5% CO_2, and EVs were harvested as described earlier with slight modifications^{14 (link),28 (link)}. Briefly, conditioned medium was aspirated and centrifuged at 300g for 10 min. Larger EVs were removed from the supernatant by two steps of centrifugation (2000g for 20 min and 12,000g for 30 min). All centrifugation steps were carried out at 4 °C. Cleared supernatants were filtered through 0.2-μm membrane filters (Thermo Fisher Scientific, USA). Smaller EVs were collected by ultracentrifugation at 100,000g for 90 min at 4 °C using Beckman L7-65 Ultracentrifuge equipped with SW 40 Ti rotor, USA. The resulting pellets, containing EVs, were resuspended in PBS and concentration of vesicles were determined using BCA assay kit (Thermo Fisher Scientific, USA).

Ukrainskaya V.M., Musatova O.E., Volkov D.V., Osipova D.S., Pershin D.S., Moysenovich A.M., Evtushenko E.G., Kulakovskaya E.A., Maksimov E.G., Zhang H., Rubtsov Y.P., Maschan M.A., Stepanov A.V, & Gabibov A.G. (2023). CAR-tropic extracellular vesicles carry tumor-associated antigens and modulate CAR T cell functionality. Scientific Reports, 13, 463.

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Purification of Influenza Viruses from Eggs

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Plaque-purified and sequenced influenza viruses were expanded in 8–10-day-old embryonated chicken eggs. Pooled allantoic fluids of approximately 20 eggs were added on top of 3 mL of a 20% sucrose solution in 0.1 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM Tris-HCl, pH 7.4, in 38.5-mL ultracentrifuge tubes (Denville). After ultracentrifugation at 112,400 × g in an L7–65 ultracentrifuge (Beckman) equipped with an SW28 rotor for 2 h at 4 °C, the pellets were recovered in 1 mL of PBS. After addition of 0.03% (v/v) formaldehyde, virus suspensions were incubated at 4 °C while shaking. After 48 h, virus suspensions were diluted with PBS and subjected to purification by ultracentrifugation as described above to remove formaldehyde. Pellets were resuspended in sterile PBS and the total protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer’s protocol.

Broecker F., Liu S.T., Suntronwong N., Sun W., Bailey M.J., Nachbagauer R., Krammer F, & Palese P. (2019). A mosaic hemagglutinin-based influenza virus vaccine candidate protects mice from challenge with divergent H3N2 strains. NPJ Vaccines, 4, 31.

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