1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Lipoid, Newark, NJ) and cholesterol (Avanti Polar Lipids, Alabaster, AL), in a DSPC/cholesterol ratio of 55/45 (mol/mol), were used as follows: lipids were dissolved at the DSPC/cholesterol 55/45 M ratio in 2,030 mL of anhydrous ethanol with heating to 40 °C, sterile filtered and then mixed with 21,000 mL of a degassed 300 mM copper sulphate solution. During this initial dispersion step, the ethanol lipid solution was maintained at ~50 °C and the copper sulphate was maintained at ~70 °C. This lipid suspension was concentrated to a final volume of 800 mL using an ultrafiltration hollow fibre cartridge system (GE Healthcare) then extruded through stacked 0.2 and 0.1 μm filters in an 800 mL thermobarrel extruder (Extruder™, Northern lipids, Vancouver, BC, Canada). The resultant nanoparticles had a mean diameter of 90–115 nm as determined by Phase Analysis Light Scattering methods (ZetaPALS, Brookhaven Instruments Corp., Holtsville, NY). To remove ethanol, the external buffer was exchanged with a 300 mM copper sulphate solution using an ultrafiltration hollow fibre column (GE Healthcare). Subsequently, the external buffer was exchanged to a sucrose/HEPES/EDTA buffer pH 7.4 (300 mM sucrose, 20 mM HEPES, 15 mM EDTA) and diluted to a final volume of 3578.0 mL to allow for irinotecan drug loading.
Cholesterol
Manufactured by Avanti Polar Lipids
Sourced in United States
Cholesterol is a lipid compound found in animal cells. It is a core component of cell membranes and is essential for various physiological processes. Avanti Polar Lipids offers high-purity cholesterol for use in research and laboratory applications.
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Lab products found in correlation
Dotap, by Avanti Polar Lipids (12 mentions)
Sodium chloride (nacl), by Merck Group (12 mentions)
Chloroform, by Merck Group (11 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (8 mentions)
Dspe peg2000, by Avanti Polar Lipids (8 mentions)
Sucrose, by Merck Group (7 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (6 mentions)
Monoolein, by Merck Group (6 mentions)
Sphingomyelin, by Avanti Polar Lipids (5 mentions)
Rockimager 1000, by Formulatrix (5 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (5 mentions)
Hepes, by Merck Group (5 mentions)
Sodium cholate, by Merck Group (5 mentions)
Cisplatin, by Merck Group (5 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine dope, by Avanti Polar Lipids (5 mentions)
Chloroform, by Thermo Fisher Scientific (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Calcein, by Merck Group (4 mentions)
Sodium diethyldithiocarbamate, by Merck Group (4 mentions)
209 protocols using cholesterol
1
Copper-Sulfate Lipid Nanoparticle Formulation
2
Lipid Nanoparticle Formulation Protocol
3
Lipid Nanoparticle Formulation Protocol
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Lipid stock solutions were formulated in chloroform. 140 μL DOPC (10 mg/mL), 30 μL DSPE-PEG(2000) maleimide (5 mg/mL), 150 μL cholesterol (5 mg/mL), and 50 μL 18:1 PEG 2000-PE (5 mg/mL, all purchased from Avanti Polar Lipids) were combined to attain a DOPC:DSPE-PEG(2000) maleimide:cholesterol:PEG 2000-PE mass ratio of 55:5:30:10 and 2.5 mg total lipid. Chloroform was evaporated under a stream of nitrogen and residual solvent was removed under vacuum overnight.
4
Lipidation and Purification of Recombinant ApoE3/4
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Recombinant (r) ApoE3 (Cat# A128, Leinco Technologies) and rApoE4 (Cat# A129, Leinco Technologies) were purchased and lipidated as previously described 79 (link). Briefly, lyophilized rApoE3/4 was solubilized in DPBS buffer with 1mM DTT and 1mM EDTA at a final concentration of 25 μM. Separately, POPC (Avanti Polar Lipids) and cholesterol (Avanti Polar Lipids) dissolved in chloroform were combined in a glass vial for a final molar ratio of ApoE:POPC:cholesterol at 1:50:10. The POPC/cholesterol mixture was dried under nitrogen until chloroform was completely evaporated. Then, DPBS was added to the dried POPC/cholesterol mixture and allowed to hydrate for 30 minutes. The mixture was added with sodium cholate (Sigma) at a 4:1 ratio (g/g) sodium cholate:POPC and incubated for 1 hour. Next, rApoE3/4 were added to sodium cholate:POPC:cholesterol mixture and incubated for 1hr at room temperature. The mixture was dialyzed in PBS at 4°C for 48 hrs using a 10,000 MWCO Slide-A-Lyzer Dialysis Cassette (Thermo Scientific) with three buffer changes. Samples were purified using a Superose 6 10/300 Increase GL column (Cytiva) with a flow rate of 0.5mL/min in PBS buffer. Samples were concentrated using an Amicon Ultra-15 10,000 MWCO concentrator (Sigma) at 4,000g for 20min at 4°C.
5
Planar Lipid Bilayer Experiments
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Planar lipid bilayer experiments were done with a vertical bilayer setup (IonoVation) as described previously (Braun et al., 2014 (link)). A 1% hexadecane solution (Merck KGaA) in n-hexane (Carl Roth) was used for pretreating the Teflon foil (Goodfellow GmbH). The hexadecane solution (ca. 0.5 µl) was added to the rim of the hole (100 µm in diameter) in the Teflon foil with a bent Hamilton syringe. The experimental solution contained 100 mM KCl and was buffered to pH 7.0 with 10 mM HEPES/KOH. For bilayer formation, we used the following lipids from Avanti Polar Lipids (Fig. S1 ): 1,2-diphythanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-diphythanoyl-sn-glycerol-3 phosphatidyl-serine (DPhPS), or n1,2-diphythanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPhPG). Cholesterol (Avanti Polar Lipids) was added to some DPhPC bilayers at a concentration of 0.15–25 mg/ml in n-pentane (Merck KGaA). Ion channel fluctuations were recorded with an EPC7 amplifier (List Medical) and digitized at 5 kHz after 1-kHz filtering with the LIH 1600 from HEKA Electronic. Data were analyzed with KielPatch (version 3.20 ZBM/2011) and Patchmaster (HEKA Electronic).
6
Lipid-Polymer Nanoparticle Formulation
7
ASCT2 Reconstitution in Liposomes
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Freshly purified ASCT2 was reconstituted in the liposomes composed of Escherichia coli polar lipids and egg phosphatidylcholine at a 3:1 ratio (w/w) and supplemented with 10% (w/w) cholesterol (Avanti Polar Lipids) 20 . For transport assays proteoliposomes were loaded with 50 mM NaCl and 10 mM or 5 mM glutamine using three freeze-thawing cycles, then
8
Porphysome Nanoparticle Synthesis and Characterization
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Porphysomes were prepared as previously described. 8 (link) Briefly, a desiccated lipid film was formed by passing a gentle stream of nitrogen gas over a suspension of lipids in chloroform [55:40:5 molar ratio of pyropheophorbide lipid (synthesized in-house): cholesterol (Avanti Polar Lipids, Alabaster, Alabama): distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (2 kDa PEG-PE, Avanti Polar Lipids)]. Residual solvent was removed from the film under vacuum for 1 h prior to rehydration with phosphate buffered saline (PBS) (150-mM NaCl, 10-mM phosphate, pH 7.4) for production of porphysome nanoparticles. The lipid suspension was subjected to five freezethaw cycles and particles formed using high-pressure extrusion (LIPEX Thermobarrel Extruder, Northern Lipids Inc, Burnaby, British Columbia, Canada) by 10 passes through double polycarbonate membranes with 100-nm pore size at 70°C. Porphysome dose for fluorescence imaging and PTT was determined using optical absorbance at 410 nm (CaryWin UV/Vis, Agilent Technologies, Mississauga, Ontario, Canada) of the porphysome lipid suspension in methanol and a previously determined molar extinction coefficient for the porphyrin-lipid (97;000 M -1 cm -1 at 410 nm). Porphysomes were prepared and stored under sterile conditions until use.
9
Giant Unilamellar Vesicle Preparation
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The lipids diphytanoylphosphatidylcholine (DiPhyPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitooleoylphosphatidylcholine (POPC), and cholesterol (Avanti Polar Lipids) were used without further purification (Fig. 1A). Fluorescent dihexadecanoylphosphoethanolamine-Texas Red (DPPE-Texas Red, Life Technologies) was included at 0.1 mol% for labeling for all membranes with DiPhyPC or POPC. Top Fluorpalmitoylundecanoylphosphatidylcholine (TopFluor-PC; Avanti Polar lipids) was included at 0.2 mol% in membranes with DOPC. Milli-Q water with a resistivity of 18 mΩ was used in all buffers. All other chemicals were purchased from Sigma Aldrich.
GUVs were created by electroformation, as described previously (28) . Briefly, lipids were combined in chloroform and dried onto conducting indium tin oxide-coated glass plates. A trimmed silicon sheet was added between the plates to form the incubation chamber. The chamber was filled with a 200 mM sucrose solution and transmitted an AC signal with Vrms 3 V at 10 Hz for 1 hr at 55 °C. The GUV solution had 13 mg of lipids per mL after electroformation. GUVs were stored at 55 °C and used within 2 days.
GUVs were created by electroformation, as described previously (28) . Briefly, lipids were combined in chloroform and dried onto conducting indium tin oxide-coated glass plates. A trimmed silicon sheet was added between the plates to form the incubation chamber. The chamber was filled with a 200 mM sucrose solution and transmitted an AC signal with Vrms 3 V at 10 Hz for 1 hr at 55 °C. The GUV solution had 13 mg of lipids per mL after electroformation. GUVs were stored at 55 °C and used within 2 days.
10
Lipid Preparation and Characterization
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All lipids, POPC, DOPC, SSM, POPC-d31, 1-palmitoyl-2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC), N-Rh-DOPE, and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). α-Spinasterol was from Tocris Biosciences (Bristol, UK). All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany) and were used without further purification.
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