Lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), were obtained from Avanti Polar Lipids (Alabaster, AL). For circular dichroism, fluorescence and 1D NMR experiments, large unilamellar vesicles were prepared using extrusion as previously described [29 (link),30 (link)]. Vesicles with zwitterionic lipids, POPC (100%) and POPE/POPC (40/60 mol%), and with negatively charged lipids, POPG/POPC (40/60 mol%) and POPA/POPC (10/90, 20/80, 30/70, 40/60 and 50/50 mol%), were produced. Lipids were dissolved in chloroform and the chloroform was evaporated overnight under nitrogen gas. The lipid mixtures were resuspended in 1 ml of the appropriate buffer for the experiment for 30 min by vortexing the samples. Next, each sample went through five cycles of freezing in liquid nitrogen and thawing in a water bath (∼60 °C). Finally, samples were extruded through a 100 nm polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL). The size of the extruded vesicles was analyzed by dynamic light scattering (ALV-laser, Langen, Germany) and vesicles were found to have a radius of around 65 nm.
Polycarbonate membrane
Manufactured by Avanti Polar Lipids
Sourced in United States
Polycarbonate membranes are thin, flat, and porous membranes made from polycarbonate material. They are primarily used as filtration and separation media in various laboratory applications.
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Lab products found in correlation
Counting beads, by Thermo Fisher Scientific (2 mentions)
Collagenase, by Thermo Fisher Scientific (2 mentions)
Lipophilic tracers, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Human cd138 microbead, by Miltenyi Biotec (2 mentions)
Streptavidin conjugation kit, by Abcam (2 mentions)
Human cytokine array q1, by RayBiotech (2 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (2 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (2 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n amino polyethylene glycol 2000 dspe peg2000, by Avanti Polar Lipids (2 mentions)
Cholesterol and chloroform, by Merck Group (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
L glutamine, by Corning (2 mentions)
Rpmi 1640, by Corning (2 mentions)
Mini extruder, by Avanti Polar Lipids (2 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphatidic acid, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphatidylethanolamine, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphatidylglycerol, by Avanti Polar Lipids (1 mentions)
29 protocols using polycarbonate membrane
1
Preparation of Lipid Vesicles for Biophysical Studies
2
Preparation of Cationic Liposomes for Delivery
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All lipids (cholesterol: 1,2-dioleoyl-3-trimethylammonium propane (DOTAP): dioleoylphosphatidylethanolamine (DOPE): 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE)-PEG = 30:50:19:1, mol: mol) were dissolved in chloroform in round flask and gently vortexed for 5 min. Chloroform was rotary-evaporated under low vacuum (500 mmHg) for 40 min at 50~60 ℃ to completely remove the organic solvent. Afterward, DDW (1.5 mL) was added to hydrate the resulting lipid film, followed by gentle vortexing until the suspension was homogenized. The suspension was passed through the polycarbonate membrane (size = 100 nm; Avanti Polar Lipids, Inc.) 11 times in an extruder (Avanti Polar Lipids, Inc.) with two 1.0 mL-glass syringes. The resulting liposomes were stored at 4℃ and used within 1 month 19 .
3
Liposome Preparation via Thin Film Hydration
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All liposomes were prepared by initially dissolving the appropriate quantity of phospholipids in chloroform and methanol to ensure the complete mixing of the components and to obtain the desired concentration. A lipid film was then formed by removing the solvent using a stream of N2 (g) followed by 3 h vacuum. To form MLVs, the dried lipids were dispersed in Tris buffer 10 mM, 150 mM NaCl, 2 mM EDTA, and thoroughly vortexed. To form LUVs, the MLVs dispersion was run through five freeze/thaw cycles and passed 11 times through a mini-extruder equipped with a polycarbonate membrane with a pore diameter of 0.1 μm (Avanti Polar Lipids, Alabaster, AL, USA).
4
Preparation of DOPS Liposomes
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The LUVs were prepared from a powder of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS, Avanti Polar Lipids, Alabaster, AL). The powder was hydrated in 1x PBS buffer and vortexed for 15 min to make a 20 mM mixture of DOPS. The mixture of lipid in buffer was passed 21 times through a mini extruder (Avanti Polar Lipids, Alabaster, AL) using a polycarbonate membrane (Pore diameter = 100 nm, Avanti Polar Lipids, Alabaster, AL). The sizes of liposomes were confirmed using dynamic light scattering. The liposomes were used within a week of their preparation in various experiments.
5
Preparation of DPPC Liposomes
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Liposomes were prepared according to the following standard procedure. 5 mg ml−1 stock solutions of lipid DPPC and 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC-d62, referred here to as d-DPPC), (Avanti Polar Lipids Inc. AL, USA) and cholesterol (Sigma-Aldrich, UK) were made in chloroform and stored at −20˚C under Argon prior to use. For DPPC liposomes a lipid film was made by adding 500 µl DPPC stock and 43 µl of cholesterol stock solution in a 10 ml round bottom flask resulting in a mol ratio of 85 : 15 mol % DPPC : cholesterol. For d-DPPC liposomes 250 µl DPPC stock and 250 µl d-DPPC stock was used, resulting in a ratio of 42.5 : 42.5 : 15 mol % // DPPC : d-DPPC : cholesterol. The chloroform was evaporated under nitrogen flow to form a thin lipid film. Lipid films were lyophilised overnight in a freeze dryer (Labconco, MO, USA) prior to rehydration. The films were hydrated with 1 ml PBS, shaken for 1 min and sonicated for 1 min. The solutions were then extruded 31 times through a polycarbonate membrane (Avanti Polar Lipids Inc. AL, USA) with a mesh size of 200 nm at 60 °C. Liposome size distribution and particle concentration were determined via NTA.
6
Preparation of DMPC Liposomal Vesicles
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The samples were prepared by the lipid film hydration method. For this, DMPC powder was dissolved in chloroform which was subsequently evaporated slowly by using a rotary evaporator. For complete chloroform evaporation, the sample was dried in an oven over night at 60 °C. The thin lipid film obtained was hydrated with an aescin solution in aqueous phosphate buffer solution. This procedure yields multilamellar vesicles (MLVs) which were enlarged with 5 freeze-thaw cycles. Small unilamellar vesicles (SUVs) were finally produced by extrusion (21×) at around 40 °C. The extruder (Avanti Polar Lipids Inc., Alabama, USA) used was equipped with a polycarbonate membrane (Whatman, Avanti Polar Lipids, Alabama, USA) with a pore diameter of 500 Å.
7
In-Vitro Antigen Release Study
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Antigen release tests were performed using custom-fabricated Franz diffusion cells (effective diffusion area: 0.785 cm2, receptor volume: 5 mL). Labeled S/O nanodispersions were prepared from PE or PE-GM labeled with FITC (FITC-PE or FITC-PE-GM). A polycarbonate membrane (Avanti Polar Lipids, Inc., Alabaster, AL, USA) was set on a cell, and the receptor compartment was filled with PBS. S/O nanodispersions loaded with FITC-PE or FITC-PE-GM were placed on the membrane and the cell was incubated for 48 h at 37 °C. Samples were collected from the receptor compartment at 1, 2, 4, 8, 24, and 48 h and replaced with the same volume of fresh media. The release of the FITC-labeled antigen was evaluated using a fluorescence spectrometer LS-55 (PerkinElmer; Waltham, MA, USA).
8
Synthesis of Gold-Loaded Liposomes
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The gold-loaded liposomes were prepared by a coextrusion method with some modification.27 (link) The mixture of HSPC, PE-NH2, and gold nanoparticles at different molar ratios was diluted to 1.0 mL in total volume using chloroform in a glass test tube, and it was vortexed gently for 10 min. Chloroform was evaporated off with a stream of Argon, and the lipid film was formed around the tube wall. This film was hydrated with 1.0 mL of RB solution (0.04 mM), followed by vigorous stirring until the suspension was homogenized. The suspension was extruded 11 times in an extruder (Avanti Polar Lipids, Inc) with two 1.0 mL glass syringes. The pore size of the polycarbonate membrane (Avanti Polar Lipids, Inc) was 100 nm. The resulting suspension was purified by centrifugation (7,000 rpm, 10 min) for three times and was stored at 4°C for further use.
9
Liposome-Mediated Protein Encapsulation and Delivery
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As previously described ( 21), the liposome was produced from 1, 2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid film. To encapsulate dye/quencher pairs, 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/pxylene-bis-pyridinium bromide (DPX) and DOPC film were rehydrated in 50 mM HEPES (pH 7.3 ) for six freeze-thaw cycles. Then, the mixture was filtered through 200 nM polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL, USA) for 20 times. After that, the redundant salt in mixture was removed in a G25 desalting column (GE Healthcare Life Sciences, Pittsburgh, PA, USA) equilibrated with 50 mM HEPES solution (pH 7.3).
The dequenching of ANTS fluorescence was measured in an ISS K2 multiphase frequency and modulation fluorometer (ISS, Champaign, IL, USA) with excitation at 380 nm and emission at 520 nm. 100 μL of liposome and 100 μg of protein were mixed with the assay buffer (150mM NaCl, 20mM Tris, pH7.4) to a final volume of 1350 μL. After 30 s of incubation, 150 μL of 1 M NaAc solution (pH 4.0) was added into the mixture to activate acidic pH-dependent membrane insertion, and the fluorescence intensity was recorded continuously for the following 180 s. The mixture was continuously stirred throughout the assay. Prior to the assay, EsxA-ST was incubated with SC-GFP (molar ratio 1:0.5) for 2 hours at RT to allow formation of the covalent bond between EsxA-ST and SC-GFP.
The dequenching of ANTS fluorescence was measured in an ISS K2 multiphase frequency and modulation fluorometer (ISS, Champaign, IL, USA) with excitation at 380 nm and emission at 520 nm. 100 μL of liposome and 100 μg of protein were mixed with the assay buffer (150mM NaCl, 20mM Tris, pH7.4) to a final volume of 1350 μL. After 30 s of incubation, 150 μL of 1 M NaAc solution (pH 4.0) was added into the mixture to activate acidic pH-dependent membrane insertion, and the fluorescence intensity was recorded continuously for the following 180 s. The mixture was continuously stirred throughout the assay. Prior to the assay, EsxA-ST was incubated with SC-GFP (molar ratio 1:0.5) for 2 hours at RT to allow formation of the covalent bond between EsxA-ST and SC-GFP.
10
Reconstitution of Ciliary Membrane Lipid Composition in Liposomes
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