Glass coverslips were cleaned with 1 M KOH for 10 min, rinsed with Milli-Q water, placed in 100% ethanol for 20 min, and plasma-cleaned for 5 min. Eight-well silicone chambers (80841; ibidi) were then attached to the cleaned coverslip. A liposome solution of 1 mg/ml with a lipid ratio of 96.5% DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 2% DGS-NTA(Ni) [2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)], 1% biotinyl-Cap-PE [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)], and 0.5% PEG5000-PE [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt)] [mole percent; all available from Avanti Polar Lipids (DOPC, 850375C), (DGS-NTA(Ni), 790404C), (biotinyl-Cap-PE, 870273C), (PEG5000-PE, 880220C)] was created by vesicle extrusion, as described in detail elsewhere (44 ). The lipid solution was added to the wells at a 1:5 ratio with Milli-Q water along with 10 mM CaCl2 for 15 min and washed repeatedly with PBS, followed by 0.5 mM EDTA in Milli-Q water to remove the excess CaCl2. The well was then washed with PBS and incubated with 1 mM NiCl2 to recharge the NTA groups. Disruption of the lipid bilayer was avoided by maintaining 100 to 150 μl of PBS in the wells.
Biotinyl cap pe
Manufactured by Avanti Polar Lipids
Biotinyl-Cap-PE is a lipid molecule used in biochemical research applications. It contains a biotin moiety covalently attached to a phosphatidylethanolamine (PE) head group. The biotin group can be utilized for various biotechnological and analytical techniques that rely on the high-affinity interaction between biotin and streptavidin or avidin.
Automatically generated - may contain errors
Lab products found in correlation
Dgs nta ni, by Avanti Polar Lipids (3 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine dopc, by Avanti Polar Lipids (2 mentions)
200 nm ptfe filter, by Merck Group (2 mentions)
Liss rhodamine, by Avanti Polar Lipids (2 mentions)
1 2 dioleoyl sn glycero 3 phospho l serine, by Avanti Polar Lipids (1 mentions)
Dgs nta, by Avanti Polar Lipids (1 mentions)
Peg5000 pe, by Avanti Polar Lipids (1 mentions)
Ninta dogs, by Avanti Polar Lipids (1 mentions)
Wha110604, by Cytiva (1 mentions)
Wha230600, by Cytiva (1 mentions)
Attofluor cell chamber, by Thermo Fisher Scientific (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphoethanolamine n cap biotinyl 16 0 biotinyl cap pe, by Avanti Polar Lipids (1 mentions)
8 protocols using biotinyl cap pe
1
Lipid Bilayer Formation on Coverslips
2
Lipid Bilayer Formation for Microscopy
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Lipid components (18:1 [Δ9-Cis] 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 18:1 1,2-dioleoyl-sn-glycero-3-phospho-L-serine [DOPS], 18:1 DGS-NTA[Ni], and 18:1 Biotinyl-Cap-PE) dissolved in chloroform were purchased from Avanti Polar Lipids and mixed at mol % indicated in the main text. The lipids were dried in round-bottom flasks under a stream of N2 for 5 min and desiccated for 2 h with house vacuum pump in a chemical fume hood. The lipid mixture was resuspended by bath sonication in 1X PBS at a final concentration of 2.5 mg/ml and extruded 10 times through a membrane with 50-nm pore size (Avanti Polar Lipids) into small unilamellar vesicles. The small unilamellar vesicle solutions were then diluted 1:1 in 1X PBS (pH 7.4) before being loaded onto the glass coverslip through the loading chamber, and incubated for 2 min to spontaneously form the lipid bilayers. The chambers were then washed with a 10X excess volume of 1X PBS. For SEM, indium-tin-oxide–coated coverslips (SPI Supplies) were used to form lipid bilayers, where DOPC was replaced with 18:1 phospho-L-serine (Kumar et al., 2009 (link)).
3
Functionalized Lipid Bilayer Platform for Immune Cell Interaction Studies
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1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (Biotinyl cap PE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (NiNTA-DOGS) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids were supplied by Avanti Polar Lipids. Planar lipid bilayers were prepared as previously described [39 (link)–41 (link), 69 (link)]. Liposomes that contained biotinyl-CAP functionalized lipids (Avanti Polar Lipids) at 4 mol% and liposomes containing NiNTA-DOGS lipids at 35 mol% were used to prepare bilayers. The final concentrations of biotinyl-CAP and NiNTA-DOGS in the bilayers were 0.01 mol% and 17.5 mol%, respectively. Streptavidin (2 μg/ml) and monobiotinylated anti-CD3 (OKT3) monoclonal antibody labeled with Alexa Fluor 488 (2 μg/ml) were reacted sequentially with the biotinylated bilayers to produce the antibody density of 50 molecules/μm2. Cy5-ICAM-1-His6 molecules were incorporated into the bilayers at the density 300 molecules/μm2. Densities of Cy5-ICAM-1 and anti-CD3 antibody on the bilayers were determined as described elsewhere [70 (link)].
4
Synthesis of Biotinylated Liposome SUVs
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SUVs were prepared using a 10 mL LIPEX Extruder (Transferra Nanosciences, Inc.). Lipids were mixed in ~500 μL chloroform with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (850375C, Avanti Polar Lipids) as the base lipid. Biotinylated lipids were incorporated at 0.05–0.2 mol% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (Biotinyl-Cap PE) (870282C, Avanti Polar Lipids). To directly tag the membrane in control experiments, N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (FITC DHPE) (23304, AAT Bioquest) was incorporated at 0.1 mol%. Lipids were dried first by rotary evaporation and second by ultrahigh purity N2. Lipid cakes were resuspended and sonicated in 3 mL nanopure water (final concentration, 2 mg mL−1) prior to performing three freeze-thaw cycles. SUVs in nanopure water were then extruded 10× through a 0.08 μm polycarbonate filter (WHA110604, Whatman) supported by a drain disc (WHA230600, Whatman). SUVs were used within ~2 weeks.
5
Preparation of Biotinylated and Fluorescent SUVs
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SUVs were prepared by rehydrating a lipid film composed primarily of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids), doped with 0.5% of Biotinyl Cap PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl), Avanti Polar Lipids), 1% DGS-Ni-NTA (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino1-carboxypentyl)iminodiacetic acid)succinyl] with nickel salt, Avanti Polar Lipids), and 0.4% LISS-Rhodamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl), Avanti Polar Lipids) in pure deionized H2O. The rehydrated solution was vortexed briefly and then sonicated using a tip-sonicator at 20% power pulsing off and on for 3 minutes. SUVs were then filtered through a 200 nm PTFE filter (Millipore). SUV solutions were stored at 4°C and used within 48 hours of preparation to avoid phospholipid oxidization.
6
Preparation of Biotinylated and Fluorescent SUVs
7
Reconstitution of β2AR in Nanodiscs
8
Supported Lipid Bilayers for Cell Adhesion
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Methodologies of supported lipid bilayer preparation and membrane functionalization have been described in Yu et al. (2011) (link) and Yu et al. (2013) (link). In brief, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) 16:0 (biotinyl-Cap-PE) were purchased from Avanti Polar Lipids, Inc. The lipids (0.2 mol% biotinyl-Cap-PE and 99.8 mol% DOPC) were mixed with an equal volume of PBS and pipetted onto cleaned glass substratum with a 25-mm coverslip placed on top for self-assembly of lipid vesicles. The lipid-coated coverslips were immersed into a deionized water bath and then placed and sealed in an Attofluor cell chamber (Thermo Fisher Scientific). The supported lipid bilayer membrane ensemble was kept under aqueous environment at all times. For membrane functionalization, the supported lipid membrane was blocked with 50 µg/ml Casein. A total of 0.1 µg/ml Cascade blue neutravidin (Thermo Fisher Scientific) was added onto supported lipid membranes, followed by 1 µg/ml biotinylated RGD, cyclo (Arg-Gly-Asp-d -Phe-Lys [Biotin-PEG-PEG]) (Peptides International). Cells were then added onto the RGD-functionalized lipid bilayer membrane and imaged or fixed within 2–3 h of preparation.
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