The HUCMSCs were subsequently seeded into a 6‐well plate at a density of 1 × 105 cells/well and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin (Gibco, Grand Island, NY) until cell confluence reached 80%. The cells were then cultured for 48 hours with Roswell Park Memorial Institute (RPMI) 1640 medium containing EV‐depleted FBS (centrifuged at 100 000 g for 18 hours). The cell supernatant was then centrifuged using Beckman Avanti Centrifuge J‐26XP (Beckman Coulter) for debris and apoptotic body removal. The supernatant was subsequently centrifuged at 110 000 g for 70 minutes (Beckman Optima L‐80 XP Ultracentrifuge with 70Ti rotor), followed by purification by centrifugation at 110 000 g for 70 minutes. All the centrifugations were conducted at 4°C. The precipitates were then resuspended in PBS and sterilized by filtration through a 0.22‐μm filter (Millipore, Darmstadt, Germany).
Beckman optima l 80 xp
Manufactured by Beckman Coulter
Sourced in United States
The Beckman Optima L‐80 XP is a high-performance ultracentrifuge designed for a wide range of applications. It is capable of achieving centrifugal forces up to 500,000 × g and can accommodate a variety of rotor types for different sample volumes and separation requirements. The Optima L‐80 XP is engineered to provide reliable and consistent performance for researchers in various fields.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using beckman optima l 80 xp
1
Extracellular Vesicle Isolation Protocol
2
Isolation and Characterization of ACC-2-derived Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ACC-2-derived MVs (ACC-2 MV) were collected from conditioned medium by differential centrifugation as described below, with a number of modifications. Briefly, the supernatant was harvested following culture for 48 h, centrifuged at 4°C sequentially at 300 × g for 10 min, 2,000 × g for 20 min and 16,500 × g for 30 min to eliminate floating cells, debris and large membrane fragments, followed by filtration through a 0.22 µm filter. MVs were pelleted by ultracentrifugation at 110,000 × g at 4°C for 70 min using a SW41 rotor (Beckman optima L-80XP; Beckman Coulter, Inc., Brea, CA, USA). The pellet was washed twice in PBS, centrifuged at 110,000 × g at 4°C for 70 min to pellet again, and then resuspended in PBS and stored the solution at −80°C until use. Total protein content of the MVs was measured using a bicinchoninic acid protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol.
3
Purification of Outer Membrane Vesicles by Histodenz Gradient
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Crude OMVs in floating buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM EDTA) containing 50% (w/v) Histodenz (D2158, Sigma Aldrich) (2.5 mL final volume) were placed at the bottom of a 13 mL centrifuge tube and overlayed sequentially with 5 mL and 2 mL of 40% and 20% Histodenz in floating buffer, respectively, and lastly with 2 mL of floating buffer with no Histodenz. The tube was centrifuged at 160,000× g, ON, in a SW 41 TI swing-out rotor (Ultracentrifuge Beckman Optima L80-XP) at 12 °C. After centrifugation, 11 fractions of 1 mL were collected from the top to the bottom of the gradient using a needle connected to a peristaltic pump. The remaining solution (~0.5 mL) was collected as fraction 12. Fractions were frozen in liquid nitrogen and stored at −80 °C.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!