Optima l 80

Manufactured by Beckman Coulter

Sourced in United States

The Optima L-80 is a high-performance ultracentrifuge designed for a wide range of applications in research and clinical laboratories. It is capable of reaching speeds up to 80,000 rpm, enabling efficient separation and analysis of microscopic cellular components and biomolecules.

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Lab products found in correlation

Biofrac fraction collector, by Bio-Rad (2 mentions) Lp data view software, by Bio-Rad (2 mentions) Biologic lp system, by Bio-Rad (2 mentions) Bca assay kit, by Thermo Fisher Scientific (2 mentions) Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions) Rnase 1, by Roche (1 mentions) Sodium dodecyl sulfate (sds), by Merck Group (1 mentions) Red safe 1x, by iNtRON Biotechnology (1 mentions) Dnase 1, by Roche (1 mentions) Proteinase k, by Roche (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Sw41ti rotor, by Beckman Coulter (1 mentions) Bradford protein assay kit, by Bio-Rad (1 mentions) Flow cytometry, by BD (1 mentions) Novaseq 6000, by Illumina (1 mentions) Histrap hp column, by Cytiva (1 mentions) Heparin hp column, by Cytiva (1 mentions) Hiload 16 600 superdex 200 column, by Cytiva (1 mentions) Polyacrylamide 6000 desalting column, by Thermo Fisher Scientific (1 mentions) Potassium bromide, by Merck Group (1 mentions)

19 protocols using optima l 80

High-Titer Bacteriophage Purification

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High-titer (1 × 10¹¹–1 × 10¹² pfu/ml in 10 mM MgSO₄) lysates were obtained from bacteriophages propagated in S. Typhimurium strain LB5000 (SGSC181; University of Calgary) and subjected to ultracentrifugation at 51,000 × g for 2 h (Optima^TM L-80; Beckman, CA, USA), as previously described24 (link). The phage titre was determined by plating serial dilutions (1:10) onto LB plates using the double agar layer method38 .

Colom J., Cano-Sarabia M., Otero J., Aríñez-Soriano J., Cortés P., Maspoch D, & Llagostera M. (2017). Microencapsulation with alginate/CaCO3: A strategy for improved phage therapy. Scientific Reports, 7, 41441.

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High-Titer Bacteriophage DNA Isolation

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High-titer (10¹¹–10¹² pfu/ml in MgSO₄ 10 mM) lysates were obtained from each bacteriophage propagated in S. Typhimurium LB5000 strain (SGSC181; University of Calgary) and by ultracentrifugation at 51,000 × g for 2 h (Optima^TM L-80; Beckman, CA, USA) (Sambrook et al., 1989 ). Bacteriophage DNA was isolated using a phenol-chloroform method (Sambrook et al., 1989 ) with slight modifications. Phage suspensions were treated with DNase I (80 U/ml; Roche Diagnostics GmbH, Germany) and RNase I (80 μg/ml; Roche Diagnostics GmbH, Germany) at 37°C for 2 h. Following the addition of 0.5% sodium dodecyl sulfate (SDS, Sigma-Aldrich, St. Louis, MO, USA) and 200 μg proteinase K (Roche Diagnostics GmbH, Germany)/ml, they were incubated at 56°C for 2 h. Phage DNA was then extracted using phenol:chloroform and precipitated with ethanol. DNA integrity was checked by using a 0.7% agarose gel electrophoresis stained with Red Safe 1X (Intron Biotechnology; Seongnam-Si, Korea); the concentration was determined in a NanoDrop ND 1000 instrument (Thermo Scientific, DE, USA).

Bardina C., Colom J., Spricigo D.A., Otero J., Sánchez-Osuna M., Cortés P, & Llagostera M. (2016). Genomics of Three New Bacteriophages Useful in the Biocontrol of Salmonella. Frontiers in Microbiology, 7, 545.

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Hematoporphyrin-loaded PGATyr Nanoparticles

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For the preparation of the hematoporphyrin-containing PGATyr-based nanoparticles (HPNPs), 10 mg of PGATyr and 5 mg of hematoporphyrin (HP) (Sigma Aldrich, UK) were dissolved in 10 mL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, UK). The resulting mixture was added drop-wise to a 5 mL polyvinyl alcohol solution (PVA, MW: 124 kDa, Sigma-Aldrich, UK) (0.5 mg/mL) and the mixture was left under constant stirring for 1 h. The mixture was then dialysed against water using dialysis tubing (MWCO: 8 kDa, Theromofisher Scientific), for 24 h. Subsequently, the suspension was centrifuged for 90 min at 38,000g, at 12 °C using a Beckman OptimaTM L-80 ultracentrifuge. The precipitated pellet was suspended in 2 mL deionised water and was probe-sonicated for 3 min, followed by further dialysis against deionised water for 12 h. The dialysed solution was then sterile-filtered using a micro syringe filter (0.22 μm, Millex GP). The suspension was snap-frozen in a sterile round-bottom flask and freeze dried for 24 h. The dried sample was resuspended in 3 mL phosphate buffered saline (PBS) and was stored at 4 °C, protected from light.

Hadi M.M., Nesbitt H., Masood H., Sciscione F., Patel S., Ramesh B.S., Emberton M., Callan J.F., MacRobert A., McHale A.P, & Nomikou N. (2021). Investigating the performance of a novel pH and cathepsin B sensitive, stimulus-responsive nanoparticle for optimised sonodynamic therapy in prostate cancer. Journal of Controlled Release, 329, 76-86.

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Microvesicle and Exosome Isolation Protocol

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Microvesicle and exosome isolation was performed as previously described with minor modifications.²² Briefly, cells were grown in FCS‐free culture medium for 24 h. The cell suspension was centrifuged at 480g at 4℃ for 5 min to remove any intact cells, followed by a 3200 g spin at 4℃ for 20 min to remove dead cells. To isolate shed microvesicles (MVs), the supernatant was centrifuged at 10,800 g at 4℃ for 20 min in an Optima L80 ultracentrifuge with a SW41Ti rotor (Beckman Coulter, Mississauga, Canada). The pellet, containing sMV, was washed once with PBS^−/− and ultracentrifuged at 10,800 g for 30 min. The pellet was dissolved in fresh medium for immediate use or stored at −80℃ for Western blot analysis. The remaining culture medium was transferred to ultracentrifuge tubes and sedimented at 110,000 g at 4℃ for at least 75 min. The supernatant constituting exosome‐free medium was removed, and the pellets containing exosomes plus proteins from media were resuspended in PBS. The suspension was centrifuged at 100,000 g at 4℃ for at least 60 min to collect final exosome pellets. The quality of exosomes was confirmed by qNano analysis (Izon instrument, UK). Protein content of the exosome pellet was quantified using the Bradford protein assay kit (Bio‐Rad, Hercules, CA).

Aschacher T., Schmidt K., Aschacher O., Eichmair E., Baranyi U., Winkler B., Grabenwoeger M., Spittler A., Enzmann F., Messner B., Riebandt J., Laufer G., Bergmann M, & Ehrlich M. (2021). Telocytes in the human ascending aorta: Characterization and exosome‐related KLF‐4/VEGF‐A expression. Journal of Cellular and Molecular Medicine, 25(20), 9697-9709.

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Milk Serum Protein Extraction Protocol

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Caseins and denatured serum proteins were precipitated by adjusting the pH of skim milk to 4.6 with 1 M HCl [21] (link), then samples were placed at 4 °C for an hour. After that, milk samples were ultracentrifuged (Optima L-80, Beckman Coulter, USA) at 33100 rpm for 90 min at room temperature to obtain milk serum samples. Based on the required sample amount and availability of samples (the amount of milk serum was limited while skim milk was available in large volumes), the protein concentration in skim milk and milk serum was determined by Kjeldahl apparatus (K1302, Sonnen Automated Analysis Instrument Co., Ltd., Shanghai, China) and Coomassie (Bradford) method, respectively.

Zhang W., Liu Y., Li Z., Xu S., Zhang J., Hettinga K, & Zhou P. (2021). Effects of microfiltration combined with ultrasonication on shelf life and bioactive protein of skim milk. Ultrasonics Sonochemistry, 77, 105668.

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Isolation of Ultrahigh-Molecular-Weight Polyethylene Particles

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Ultra‐pure water was added to fill each ultracentrifugation tube and the tubes were centrifuged at 125,755 g at 20°C for 3 h (Optima L80 ultracentrifuge, Beckman Coulter, USA). All materials with a density greater than water (~1.0 g cm⁻³) which included: proteins, metal, calcium phosphate, bone cement, extracellular matrix, and high‐density lipids, were pelleted. The supernatant contained materials with a density less than water such as UHMWPE wear particles and lipids. The supernatant for each sample was decanted 5 ml at a time into clean glass universals ready for process 1. The pellet of particles remaining in the ultracentrifuge tube was continued to process 2.

Stratton‐Powell A.A., Williams S., Tipper J.L., Redmond A.C, & Brockett C.L. (2022). Mixed material wear particle isolation from periprosthetic tissue surrounding total joint replacements. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 110(10), 2276-2289.

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Isolation and Characterization of Endothelial Progenitor Cell-Derived Microvesicles

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EPCs at 80% confluence were cultured in serum-free medium for 24 h; the culture medium was collected and centrifuged at 4°C (300× g for 5 min followed by 2,000× g for 15 min). The supernatant was ultracentrifuged at 100,000× g (Optima L-80; Beckman Coulter, Brea, CA, USA) for 2 h at 4°C, and resuspended in PBS followed by ultracentrifugation at 100,000× g for 1 h at 4°C. EPC-MVs were characterized by Transmission electron microscopy (TEM) and flow cytometry analysis. For TEM, MVs were fixed in Karnovsky fixative, dehydrated in alcohol, dried on a glass surface, and sputter-coated with gold. The specimens were visualized using an FEI Tecnai-10 transmission electron microscope (Philips, Amsterdam, The Netherlands). For flow cytometry analysis, EPC-MVs were resuspended and incubated for 30 min at 4°C in the dark with phycoerythrin (PE)-conjugated antibodies against CD34 (1:50; cat. no. sc-7324) and FITC-conjugated antibodies against annexin V (1:100; cat. no. sc-74438; all from Santa Cruz Biotechnology). An isotype-matched (IgG) nonspecific antibody served as a negative control. After incubation, labeled MVs were washed with PBS three times, resuspended with 70 μl of PBS, and analyzed by flow cytometry (BD Biosciences).

Zeng W., Lei Q., Ma J., Gao S, & Ju R. (2021). Endothelial Progenitor Cell-Derived Microvesicles Promote Angiogenesis in Rat Brain Microvascular Endothelial Cells In vitro. Frontiers in Cellular Neuroscience, 15, 638351.

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Nucleosome Purification and Fixation via GraFix

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The reconstituted nucleosomes were purified and fixed using the
GraFix method. Briefly, a sucrose gradient was created using 2.3 ml of a 20
% sucrose solution containing 15 mM HEPES [pH 7.4] and 4 % paraformaldehyde
aqueous solution (EM Grade) (Electron Microscopy Sciences) and 2 ml of a 10%
sucrose solution containing 15 mM HEPES [pH 7.4]. Reconstituted nucleosome
samples were overlaid onto the sucrose gradient and spun at 32000 rpm for 20
h at 4 °C using SW55Ti rotor in Optima L80 (Beckman Coulter).
Fractions containing the fixed samples were collected and dialyzed in 15 mM
HEPES (pH 7.4) for 48 h at 4 °C with one fresh exchange of buffer
halfway through, using a Tube-O-DIALYZER (50 kDa) (G-Biosciences). Samples
were concentrated using an Amicon Ultra 100K centrifugation filter
(Millipore) to final concentration of 247 ng/μl DNA.

Arimura Y., Shih R.M., Froom R, & Funabiki H. (2021). Structural features of nucleosomes in interphase and metaphase chromosomes. Molecular cell, 81(21), 4377-4397.e12.

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Polysome Profiling of mTORC1 Regulation

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Polysome profiling was performed as described with modifications^{13 (link)}. Briefly, cells were treated with 100μg/ml cycloheximide (CHX) for 5min, harvested, washed twice with PBS containing 100μg/ml CHX, and centrifuged at 300×g for 5min at 4^oC. Cells were respuspended in 425μl hypotonic buffer (5mM Tris-HCl pH7.5, 2.5mM MgCl2, 1.5mM KCl, protease inhibitor cocktail) and transferred to a pre-chilled 1.5ml tube. Then 5μl of 10mg/ml CHX, 1μl of 1M DTT, and 100U RNase inhibitors were added to cells followed by vortexing for 5sec. Then 25ul of 10% Triton X-100 and 25μl of 10% sodium deoxycholate were added to cells followed by vortexing for 5sec. The supernatant were transferred to a pre-chilled 1.5ml tube after centrifugation at 21,000×g for 5min at 4^oC. The same OD amount of lysates from each sample were loaded onto a 10-50% sucrose gradient, and centrifuged at 35,000rpm for 2h at 4^oC using SW40Ti rotor in a Beckman Coulter Optima L-80 ultracentrifuge with no brake. Samples were analyzed on the Bio-Rad BioLogic LP system and BioFrac fraction collector, and chased with 60% (w/v) sucrose with bromophenol blue at 1ml/min. Data were analyzed using the Bio-Rad LP Data View software. Polysome fractions were collected at 0.5 ml/fraction. For mTORC1 inhibition, cells were treated with DMSO or 2.5μM PP242 for 12h.

Liu X., Zhang Y., Ni M., Cao H., Signer R.A., Li D., Li M., Gu Z., Hu Z., Dickerson K.E., Weinberg S.E., Chandel N.S., DeBerardinis R.J., Zhou F., Shao Z, & Xu J. (2017). Regulation of Mitochondrial Biogenesis in Erythropoiesis by mTORC1-Mediated Protein Translation. Nature cell biology, 19(6), 626-638.

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Measuring Drug Encapsulation Efficiency

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To determine drug encapsulation efficiency, nanoemulsion formulation samples (1 mL) were added to Eppendorf centrifuge tubes, then subjected to ultracentrifugation (Beckman Optima L-80, Beckman, Brea, CA, USA) at 13,000 × g for 2 hrs to obtain a clear supernatant.21 (link) The supernatant solution (0.1 mL) was withdrawn and stored at 4 °C until analysis. The supernatant was diluted with a 1:1 (v/v, 2.9 mL) methanol:water mixture to determine RIF content, and %EE was calculated using the following equation (3):

(3)

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$${\rm{EE}}\left({\rm{\% }} \right){\rm{\ =\ }}\left({{{\rm{E}}_{\rm{2}}}{\rm{/}}{{\rm{E}}_{\rm{1}}}} \right){\rm{ \times 100}}$$
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where E₁ and E₂ were the total drug content added to the formulation and the total free amount of drug, respectively. Rifampicin content was analyzed using a UV-Vis spectrophotometer at 337 nm.

Hussain A., Altamimi M.A., Alshehri S., Imam S.S., Shakeel F, & Singh S.K. (2020). Novel Approach for Transdermal Delivery of Rifampicin to Induce Synergistic Antimycobacterial Effects Against Cutaneous and Systemic Tuberculosis Using a Cationic Nanoemulsion Gel. International Journal of Nanomedicine, 15, 1073-1094.

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