As reported in previous studies, we have prepared RSL3@O2-ICG NBs using the thin-film hydration ultrasound method.44 (link) First, mix DSPC and DSPE-PEG-2000 at a mass ratio of 9:1, and then add 10ul of 5 mM RSL3 in methylene chloride and methanol (2:1, vol/vol). After entirely dissolving and mixing, transfer the above solution to a beaker and evaporate to form a lipid film. The dried film was hydrated into a lipid suspension with 5mL of 100ug/mL ICG in PBS. Next, the suspension was extruded 20 times through a 200nm membrane through a micro-extruder (Avanti Polar Lipids, Alabaster, AL). Then, the squeezed suspension was transferred to a sealed vial, and the syringe was evacuated and replaced with pure oxygen. The mixture was then mechanically shaken in a dental mixer (YJT Medical Apparatus and Instruments, Shanghai, China) for 60 seconds and resuspended in 2 mL of PBS solution for storage at 4°C. All processes are carried out in a dark environment. The blank nanobubbles (without RSL3 or ICG) were prepared using the same method. The stability of the RSL3@O2-ICG NBs and blank nanobubbles was evaluated by a hemocytometer at different time points, such as 6 h, 12h, 24h, and 48h after preparation at 4°C.
Microextruder
Manufactured by Avanti Polar Lipids
Sourced in United States
The Microextruder is a laboratory instrument designed for the extrusion of small samples. It is used to produce uniform and reproducible lipid vesicles or liposomes through a membrane with defined pore size. The Microextruder operates at controlled temperatures to enable the extrusion of temperature-sensitive materials.
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Lab products found in correlation
5 protocols using microextruder
1
Synthesis of RSL3@O2-ICG Nanobubbles
2
Surface Plasmon Resonance Lipid Vesicle Binding Assay
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Lipids dissolved in chloroform were dried under nitrogen and resuspended in 500 μl 20 mM Tris-HCl, 500 mM NaCl, pH 7.4. Mixture was vortexed for 25 min at room temperature and sonicated for 1 min in a Branson 1,200 sonifier. Large unilamellar vesicles with 100-nm diameter were prepared with a microextruder (Avanti Polar Lipids) using a 100-nm polycarbonate filter. SPR measurements were performed at 23 °C using lipid-coated L1 chip in the BIACORE X system56 (link). Briefly, after washing the sensor chip with buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl), PC/PS/PtdIns3P (or PtdIns4P) (77:20:3) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC; Avanti Polar Lipids) vesicles were injected at 5 ml min−1 to the active and the control surface, respectively. Assuming a Langmuir-type binding between the protein (P) and protein binding sites (M) (that is, P+M↔PM), Req values were plotted versus P0, and the Kd value was determined by a nonlinear least-squares analysis of the binding isotherm using an equation, Req=Rmax/(1+Kd/P0) (ref. 40 (link)). Each data set was repeated three times for statistical significance.
3
Platelet Isolation and Extruded Nanovesicle Production
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Platelets were isolated from the blood of volunteers according to reported protocols (11 (link)). The whole sample of fresh blood was centrifuged at room temperature at 100 × g for 20 min. After centrifugation, the whole blood sample was divided into three layers, from top to bottom: the plasma layer, white blood cell layer, and red blood cell layer. The pale-yellow plasma layer was collected and centrifuged at 800 × g for 20 min. After centrifugation, the pellets were platelets. The pellet was resuspended in 1 μM PGE1 in phosphate buffered saline to inhibit platelet aggregation and stored at room temperature until use. The platelet solution of 1 × 106 cells/mL was squeezed six times with 400nm, 200nm, and 100nm pore size polycarbonate membrane filters (Whatman) in a micro extruder (Avanti Polar Lipids), and NPVs were obtained after centrifugation.
4
Preparation and Characterization of LUVs
5
Liposome Preparation and Characterization
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The extrusion method was used to obtain liposomes (large unilamellar vesicles or LUV). For this purpose, 7.5 mg of dry lecithin was incubated for several hours in 0.75 mL of the following buffer: 10 mM Tris/HCl (pH 8.5), 50 mM SRB and 50 μM EGTA. Five freezing/thawing cycles were performed before the multilamellar liposome suspension was extruded 11 times through a 0.1 μm polycarbonate membrane using a microextruder (Avanti Polar Lipids, Birmingham, AL, USA). After extrusion, liposomes were applied on a Sephadex G-50 column to remove the external SRB. The release of SRB from liposomes incubated in 10 mM Tris/HCl (pH 7.5), 50 μM EGTA, and 40 mM KCl buffer was evaluated by the increase in SRB fluorescence using an Ocean Optics FLAME-T-UV-VIS fiber-optic system (Ocean Optics Inc., Dunedin, USA), as described earlier [21 (link)]. A 0.1% solution of Triton X-100 was added to release all of the loaded SRB.
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