Synthetic lipid components, including 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero3-(phospho-L-serine) (DOPS), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (PEG-PE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE), were purchased from Avanti Polar Lipids (USA). As indicators in the lipid mixing assay, the lipid analog fluorescent dyes DiI and DiD were used as FRET donor and acceptor dyes, respectively (Invitrogen, USA). Chloroform stock solutions were mixed, and the chloroform was removed under vacuum in a desiccator for 16 h. The molar ratio of POPC:DOPS:PEG-PE was 92:7:1, and 10‐5 mol % Rhod-PE was added for the formation of the supported lipid bilayer (SLB). For an in vitro lipid mixing assay, the molar ratio of POPC:DOPS:Chol:DiI (or DiD) was 71:7:20:2. The lipid mixtures were then rehydrated in 25 mM HEPES buffer with 100 mM KCl (pH 7.4), and ten freeze-and-thaw cycles were performed using liquid nitrogen and a water bath at 37°C. The solution was passed through a mini extruder (Avanti Polar Lipids) ten times with a 100 nm polycarbonate filter (Whatman, UK). Next, extrusion was performed to obtain monodisperse unilamellar vesicles using a mini extruder (Avanti Polar Lipids) with a 100 nm polycarbonate filter (Whatman).
Mini extruder
Manufactured by Avanti Polar Lipids
Sourced in United States, United Kingdom, Italy
The Mini-extruder is a compact and versatile laboratory device designed for the extrusion of lipid vesicles and liposomes. It features a manual operation mechanism that allows for controlled and reproducible extrusion of samples through polycarbonate membranes with defined pore sizes.
Automatically generated - may contain errors
Lab products found in correlation
Polycarbonate membrane filter, by Cytiva (8 mentions)
Polycarbonate filter, by Avanti Polar Lipids (6 mentions)
Zetasizer nano z, by Malvern Panalytical (5 mentions)
Cholesterol, by Merck Group (5 mentions)
Chloroform, by Avanti Polar Lipids (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Polycarbonate porous membrane, by Cytiva (4 mentions)
Polycarbonate membrane, by Cytiva (4 mentions)
Tecnai g2 transmission electron microscope, by Thermo Fisher Scientific (3 mentions)
Zetaview system, by Particle Metrix (3 mentions)
Basic fibroblast growth factor (bfgf), by Applied Biological Materials (3 mentions)
Epidermal growth factor (egf), by Applied Biological Materials (3 mentions)
Ren cells, by Merck Group (3 mentions)
Accutase, by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Thermomixer, by Eppendorf (3 mentions)
Pd 10 desalting column, by GE Healthcare (3 mentions)
Bio beads sm 2, by Bio-Rad (3 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phospho l serine, by Avanti Polar Lipids (3 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine popc, by Avanti Polar Lipids (3 mentions)
401 protocols using mini extruder
1
Preparation of Lipid Vesicles for In Vitro Assay
2
Isolation of Cell Membrane Nanovesicles
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The cell membrane was collected by a repeated freeze‐thaw process as previously reported.[9 ] Cell suspensions in phosphate buffered saline (PBS) were frozen at ‐80 °C, thawed at room temperature, and pelleted by centrifugation at 1000 g for 5 min. Following three repeated washes with cold PBS mixed with protease inhibitor cocktail (#87785, Thermo Scientific), cell membranes were suspended in cold PBS and sonicated in a sterile 1.5 mL EP tube under lower power (22.5 W, 20 s ×3) on ice. Then the vesicles were isolated by multi‐steps of differential centrifugation before being resuspended in PBS. The product was then introduced to a Mini‐Extruder (Avanti Polar Lipids) equilibrated in PBS (200 nm pore‐sized membrane filters) to further purify uniform nanovesicles. Vesicles were stored at 4 °C for 1 week or placed in long‐term storage at ‐80 °C. Total membrane protein content was quantified by a BCA protein assay kit (#23227, Thermo Scientific). For CoVR‐MV generation, hACE2‐MVs and hDPP4‐MVs were mixed and then extruded through 200‐nm pores on the Mini‐Extruder (Avanti Polar Lipids).
3
Preparation of Calcein-Loaded LUVs
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LUVs were prepared by the extrusion method as described previously (Rekdal et al. 2012 (link), Arias et al. 2014a) . Briefly, phospholipids dissolved in chloroform and methanol (2:1) were combined in a glass vial, dried under a gentle stream of nitrogen gas, and then placed under vacuum overnight to remove all trace of the organic solvents. The dry lipid films were hydrated with 70 mM calcein dissolved in leakage buffer (HEPES 10 mM, NaCl 150 mM and EDTA 1mM, pH 7.5) and multilamellar lipid vesicles (MLVs) were formed by vigorous vortexing. The MLVs suspension was then freeze-thawed seven times with liquid nitrogen and warm water (~ 40°C). LUVs (100 nm in diameter) were prepared from the MLVs suspension by the extrusion method using a mini-extruder (Avanti lipids, Inc.) and 0.1 µm polycarbonate filters (Nucleopore Filtration Products, Pleasonton, CA). Free calcein was removed from the LUVs suspension by size exclusion chromatography on a Sephadex G-50 column previously equilibrated with leakage buffer.
4
Fabrication of Coumarin-Labelled PLGA Nanoparticles
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P@R837 NPs were fabricated as previously reported 5 . Briefly, PLGA (25 mg/mL) was dissolved in CH2Cl2 with 200 μg R837 (in DMSO at 5 mg/mL), then 0.5 mL polyvinylpyrrolidone (PVP) solution (5 % w/v) was added into the PLGA mixture followed by tip-sonication (40 % power for 3 minutes, 3 seconds on and 1 second off). After that, 2.5 mL PVP solution (5% w/v) was added dropwise into the obtained solution under constant stirring overnight at room temperature. The P@R837 NPs were obtained after centrifugation at 3,500 g for 25 min. The naked PLGA NPs (denoted as P) were fabricated using the same protocol, without adding the R837. To prepare CCMP@R837, 4T1 cell membrane pellet was bath-sonicated and physically co-extruded with P@R837 through a 200 nm polycarbonate membrane (Avanti Lipids) by mini extruder (Avanti Lipids) for 11 passes. Fluorescence labelled P, P@R837 and CCMP@R837 were named as F#P, F#P@R837 and F#CCMP@R837. These NPs were prepared by adding coumarin 6 into the solution of PLGA.
5
Liposome Preparation for ER Targeting
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ER targeting liposomes were prepared as described.52 (link),53 (link) Briefly, 1 μmol of total phospholipids was mixed with the molar ratio as indicated: control (No PE or PC), PI:PS= 0.2 μmol: 0.2 μmol; low PE, PE:PC:PI:PS= 0.1 μmol: 0.5 μmol: 0.2 μmol:0.2 μmol; high PE, PE:PC:PI:PS= 0.5 μmol: 0.1 μmol: 0.2 μmol: 0.2 μmol, dried down and vacuumed for 30 min. The dried phospholipid mixture was rehydrated in 1ml of PBS at 55°C for 30 min with vigorous vortex. The solution was then passed through a 0.1 μm polycarbonate membrane 10 times using a mini-Extruder (Avanti lipids, Cat # 610000) to form unilaminar liposomes.
6
Large Unilamellar Vesicle (LUV) Preparation
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LUVs (Large Unilamellar Vesicles) were prepared as previously reported.26 (link) Briefly, the lipids in chloroform were dried under nitrogen and then overnight under high vacuum. The lipid film was then resuspended in 1 mL of buffer (20 mM phosphate buffer at pH 7.4 with 150 mMNaCl and 0.8 mM EDTA), subjected to 5 freeze–thaw cycles and vortexed. LUVs were prepared by extrusion through a polycarbonate membrane with an Avanti Polar mini-extruder (20 times through two-stacked polycarbonate membranes with pore sizes of 100 nm). LUVs were composed of 100% POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and of a 70/30 (w/w) combination of POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol).
7
Preparation of Large Unilamellar Vesicles
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LUVs (Large Unilamellar Vesicles) were prepared as previously reported [29 (link)]. Lipids were dissolved in chloroform/methanol 9:1. The solvent was then removed by rotary evaporation and then overnight under high vacuum. The lipid film was then hydrated by adding 1 mL of buffer (20 mM phosphate buffer at pH 7.4 with 150 mM NaCl and 0.8 mM EDTA) and then subjected to 5 freeze-thaw cycles and vortexed. The multilamellar vesicles (MLVS) formed were extruded through a polycarbonate membrane with pore size of 100 nm using a mini extruder (Avanti Polar). The obtained LUVs were composed of 100% POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and of a 70/30 (w/w) combination of POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol). The final concentration of the lipid was 10 mM.
8
Biophysical Characterization of Lipid Vesicles
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Proton and carbon nuclear magnetic resonance spectra (1H, 13C NMR) were recorded on a Bruker Avance 400 MHz/600 MHz spectrometer. Mass spectra were recorded on a Micromass GCTTM and a Micromass LCTTM. Fluorescence measurements were performed on a Varian Cary Eclipses fluorescence spectrometer equipped with a stirrer and a temperature controller (kept at 25°C unless otherwise noted). A Mini-Extruder used for the preparation of LUVs was purchased from Avanti Polar lipids. The size of EYPC vesicles was determined using a Delsa™ Nano Submicron Particle Size and Zeta Potential Particle Analyzer (Beckman Coulter Inc., USA). Preparative reverse phase HPLC was performed using a Waters 2454 Multisolvent Delivery System with a Waters 2489 UV/visible detector operating at 254 nm.
9
Preparation and Characterization of Lipid Vesicles
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Vesicles were prepared by drying desired amount of lipids from stock solutions in chloroform/methanol using nitrogen stream and then were kept for 5–6 hrs in vacuum to remove any traces of organic solvents. For preparation of large unilamellar vesicles (LUVs), desiccated lipid film was hydrated in 10 mM sodium phosphate buffer (pH 7.4) and kept at 4°C overnight. Multilamellar vesicles (MLVs) were formed by vortexing lipid suspensions in sodium phosphate buffer for several minutes. MLVs were passed several times through polycarbonate membranes of 200 nm pore size using mini-extruder (Avanti Polar Lipid Inc.) to get LUVs [42 (link)]. Homogeneous size distribution around 200 nm was confirmed by dynamic light scattering analysis using a Photocor Complex-dynamic light scattering (DLS) instrument (Photocor Instruments, MD). A laser of wavelength 632.8 nm was used for collecting the data. The data were processed using DynaLS software (V.2.8.3). GUVs doped with 0.1 mol% Rh-PE were prepared using swelling method [43 (link)]. The desiccated lipid film was hydrated with 10 mM sodium phosphate buffer, pH 7.4 at 45°C for 15 mins followed by 10 hrs incubation at 60°C to get vesicles.
10
Preparation and Characterization of Liposomes
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Liposomes of the appropriate size were prepared for different experiments. An appropriate amount of lipids was weighed and dissolved in CHCl3/CH3OH (70/30 v/v) mixture to prepare liposomes at different composition. Typical mixtures volumes were 2 mL of a lipid concentration range of 0.1–1 mM. In these conditions fully hydrated liposomes were obtained. A thin film of lipids was obtained through evaporation of the organic solvent with dry nitrogen gas and vacuum desiccation. Lipid films were kept in vacuum overnight to remove all residual organic solvent, then hydrated with a definite amount of PBS buffer pH 7.4 and finally vortexed to obtain a suspension of Multi Lamellar Vesicles (MLVs). In addition, Large Unilamellar Vesicles (LUVs) were prepared by extrusion using a Mini-Extruder (Avanti Polar Lipid Inc.) according to method described in Hope et al.26 (link). In fact, the MLVs suspension was freeze-thawed six times then passed through a 100 nm pore size polycarbonate membrane 25 times and the obtained LUVs suspensions were used to study the binding with peptides. Dynamic light scattering measurements were performed to check the size of the vesicles after the extrusion protocol.
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