Micelles containing SPIONs were synthesized using a previously reported protocol.59 (link) Briefly, 4 mg Poly(ethylene oxide)-b-poly(ε-caprolactone) 4.0-b-3.0 (PEG-PCL 4k:3k) (Polymer Source), 0.4 mg PCL-5k, and 0.6 mg SPIONs were dissolved in 200 μL toluene. The solution was added to 4 mL of water and sonicated to form a homogeneous emulsion, and the toluene was allowed to evaporate at room temperature overnight to form micelles. The product was concentrated using a 100 kDa centrifugal filter (Millipore) and purified by centrifugation at 3,000 g × 10 minutes to remove large aggregates followed by centrifugation at 16,100 g × 10 minutes to remove empty micelles. Micelles were imaged by transmission electron microscopy (TEM) using a JEOL-1010 system and characterized by dynamic light scattering (Malvern). The micelles’ magnetic properties were measured by NMR (Bruker).
100 kda centrifugal filter
Manufactured by Merck Group
Sourced in United States
The Merck 100 kDa centrifugal filter is a laboratory device used for separating and concentrating molecules based on their molecular weight. It operates by applying centrifugal force to push a sample solution through a semi-permeable membrane, allowing molecules smaller than 100 kDa to pass through while retaining larger molecules.
Automatically generated - may contain errors
3 protocols using 100 kda centrifugal filter
1
Micelle-Encapsulated Superparamagnetic Iron Oxide Nanoparticles
2
Isolation of Bacterial Extracellular Vesicles
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The extracellular vesicles (EVs) were isolated based on the ultracentrifugation method. GAS strains were grown to the early-stationary phase (O.D.600 = 1.0) or cultured for 8–16 h at 37ºC with 5% CO2 supplementation. The bacterial culture supernatants were collected, and bacterial cells were removed by utilizing an 0.22 µm filter (Millipore; MA, USA). The cell-free culture supernatant, with or without 100 kDa centrifugal filter (Millipore) concentrated, was centrifugated at 3300 ×g at 4ºC for 10 min to remove the debris. Finally, the supernatant was centrifugated at 175,000 ×g at 4ºC for 4 h. The resulting pellet (EVs) was resuspended by sterilized 1× PBS and stored at −80ºC before analysis.
3
Exosome Mimetics Isolation Protocol
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All the cells were incubated at 37°C with 5% CO2, and hUMSCs were grown in a mesenchymal stem cell basal medium (Cell Farm, Shanghai, China) with 10% exosome-free fetal bovine serum (FBS) (Gibco). FBS was managed by ultracentrifugation at × 120,000 g for 120 min to remove the exosomes from FBS. All of the supernatant was collected from hUMSCs from passages 4 to 8. After collecting the supernatant of hUMSCs, the exosomes were ultracentrifuged at ×300 g for 10 min, ×2000 g for 10 min, and ×15,000 g for 40 min to remove cells and cell debris. Next, the supernatant was ultra-centrifuged at ×1,00,000 g for 70 min twice in Ultra-Clear centrifuge tubes (Beckman Coulter, United States) for exosome purification. Finally, the precipitate was resuspended in PBS and stored at −80°C for identification and the following experiments.
After hUMSCs reached a confluence of approximately 70%–80%, the cells were resuspended in a PBS solution of 1 × 106 cells per 1 ml. The EMs were collected using serial extrusive approaches through polycarbonate membranes (Whatman, United States) of 10 nm, 5 nm, 1 nm, 800 μm, 400 μm, and 200 μm using a mini-extruder (Morgec, Shanghai, China). Exosome mimetics were centrifuged at × 100,000 g for 70 min twice and purified by a 100 kDa centrifugal filter (Millipore, CA, United States) at ×4,000 g for 20 min. The final EM sample was stored at −80°C.
After hUMSCs reached a confluence of approximately 70%–80%, the cells were resuspended in a PBS solution of 1 × 106 cells per 1 ml. The EMs were collected using serial extrusive approaches through polycarbonate membranes (Whatman, United States) of 10 nm, 5 nm, 1 nm, 800 μm, 400 μm, and 200 μm using a mini-extruder (Morgec, Shanghai, China). Exosome mimetics were centrifuged at × 100,000 g for 70 min twice and purified by a 100 kDa centrifugal filter (Millipore, CA, United States) at ×4,000 g for 20 min. The final EM sample was stored at −80°C.
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