Sorvall wx ultra 80

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Sorvall WX Ultra 80 is a high-speed centrifuge designed for laboratory use. It is capable of reaching speeds up to 80,000 rpm and generating forces up to 500,000 x g. The centrifuge can accommodate a variety of rotor types and sample volumes, making it suitable for a wide range of applications.

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Lab products found in correlation

Th 660 rotor, by Thermo Fisher Scientific (2 mentions) Optipreptm density gradient medium, by Merck Group (1 mentions) Trypan blue, by Merck Group (1 mentions) Vivaspin, by Sartorius (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Cytoflex s, by Beckman Coulter (1 mentions)

4 protocols using sorvall wx ultra 80

Niosome Characterization and Encapsulation

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The size and morphology of niosomes were also determined by negative staining-transmission electron microscopy. After niosomes were negatively stained, their size and morphology were observed using a TecnaiTM G2 Spirit Twin transmission electron microscope (FEI, Hillsboro, OR, USA). Ultracentrifugation was adopted for the separation of free and entrapped drug. The niosomal suspension was ultracentrifuged at 20,000 rpm at 4°C for 1 hour in an ultracentrifuge (Sorvall WX Ultra 80; Thermo Fisher Scientific, Waltham, MA, USA) in order to separate the incorporated drug from the free form. The precipitate of niosomes was dissolved in 20% Triton X-100 in methanol. The drugs in the supernatant and precipitate were analyzed by HPLC to determine the encapsulation percentage. The entrapment capacity of niosomes were calculated as,
where T is the total amount of drug that is detected both in the supernatant and sediment, and C is the amount of drug detected only in the supernatant.

Song Q., Li D., Zhou Y., Yang J., Yang W., Zhou G, & Wen J. (2014). Enhanced uptake and transport of (+)-catechin and (−)-epigallocatechin gallate in niosomal formulation by human intestinal Caco-2 cells. International Journal of Nanomedicine, 9, 2157-2165.

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Extracellular Vesicle Isolation via Iodixanol Gradient

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Flotation in an iodixanol gradient was performed as described in Crescitelli et al. (2020 (link)) with some modifications. The 100K pellet obtained after differential centrifugation was re‐suspended in 1 mL of 40% iodixanol (v/v) (OptiPrep^TM Density Gradient Medium, Sigma) in PBS and bottom loaded. For the discontinuous iodixanol gradient equal volumes of solutions of 30%, 20% and 10% iodixanol were layered on the top of the sample (Figure 6a) and centrifuged in a 4‐mL tube (Beckman Coulter, 328874) at 180,000 × g for 19h, at 0°C (Sorvall WX‐Ultra 80, Thermo Fisher Scientific) in a TH‐660 rotor (Thermo Fisher Scientific). Fractions of 500 μL from top to bottom were collected from the tube. An opaque band of EVs was recovered in fraction 6.

Ditte Z., Silbern I., Ditte P., Urlaub H, & Eichele G. (2022). Extracellular vesicles derived from the choroid plexus trigger the differentiation of neural stem cells. Journal of Extracellular Vesicles, 11(11), 12276.

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Isolation of Extracellular Vesicles from Cells

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Cells with a confluency of 70%–80% were washed twice with PBS. To exclude carrying along EVs from serum, Z310 cells and MEFs were maintained for 2 days in serum‐free conditioned medium. The medium was then aspirated without disturbing the cells and subjected to differential centrifugation, as described by Kowal et al. (2016 ) (Figure 2a). The percentage of live cells at the time of harvesting was determined by Trypan Blue (Sigma). The cell pellet was collected and resuspended in 0.4% (v/v) Trypan Blue. Dead and live cells were counted using a hemocytometer (Nexcelon, Bioscience). Greater than 90% of the cells were viable, for example, in the case of Z310 cells, viability was 92.5± 2.1%. Centrifugation steps (Figure 2a): 300 × g for 10 min to remove cells and cell debris, then at 2000 × g for 20 min (Eppendorf 5702R) and 10,000 × g for 40 min (Eppendorf 5417R) to remove larger vesicles. The 10K supernatant was concentrated at 0°C using Vivaspin (300000 MWCO, Sartorius, Göttingen, Germany). This concentrated supernatant was centrifuged at 100,000 × g, for 60 min (Sorvall WX‐Ultra 80, Thermo Fisher Scientific, USA) in a TH‐660 rotor (Thermo Fisher Scientific). The resulting pellet was re‐suspended in ice‐cold PBS and centrifugation was repeated under the same condition. The resulting pellet was re‐suspended in PBS and stored at −80°C.

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Evaluating Mesenchymal Stem Cell Immunosuppression

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MSCs were plated at 20,000 cells/cm 2 in 48 well plates in complete medium and allowed to adhere for 24 hours. The cells were then subjected to priming as previously described, followed by two PBS -/-washes.
PBMCs were labeled with CFSE (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions and 500,000 PBMCs were added to each well. After the addition of PBMCs, stimulating anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA) were added at 500,000 each per well, and 10 9 EVs were added to the appropriate wells.
The assays took place in complete RPMI medium formulated as above, but with EVdepleted FBS. EVs were depleted by centrifuging FBS at 100,000g for 1 hour at 4°C (Sorvall WX Ultra 80, Thermo Fisher Scientific, Waltham, MA) and using the supernatant All flow analysis was performed using a CytoFLEX S (Beckman Coulter, Hialeah, Florida), with 20,000 events collected per sample for the uptake and immunosuppression assays (S3). All data was analyzed using FlowJo software (Treestar, Inc., Ashland, Oregon).
Cellular debris, activating beads, and doublets were gated out via scatter properties. T cell proliferation was calculated according to the formula below, where MI is the median fluorescence intensity of CFSE stained samples, and PS is the proliferation score (32, 33) :

Andrews S.H., Maughon T.S., Marklein R.A., & Stice S.L. (2020). Priming of MSCs with inflammation-relevant signals affects extracellular vesicle biogenesis, surface markers, and modulation of T cell subsets.

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