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.
Peg5000 pe
Manufactured by Avanti Polar Lipids
PEG5000-PE is a lipid product provided by Avanti Polar Lipids. It consists of a polyethylene glycol (PEG) moiety covalently linked to a phosphatidylethanolamine (PE) headgroup. The core function of this product is to serve as a hydrophilic polymer-lipid conjugate for use in various research and development applications.
Automatically generated - may contain errors
Lab products found in correlation
Hellmanex, by Merck Group (2 mentions)
Dgs nta ni, by Avanti Polar Lipids (2 mentions)
Biotin dppe, by Avanti Polar Lipids (2 mentions)
Biotinyl cap pe, by Avanti Polar Lipids (1 mentions)
Dgs nta, by Avanti Polar Lipids (1 mentions)
Dspe peg2000 biotin, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 oleoyl glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (1 mentions)
7 protocols using peg5000 pe
1
Lipid Bilayer Formation on Coverslips
2
Preparation of Ni2+ Decorated SUVs
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POPC (Avanti Lipids, Cat# 850457P), DGS-NTA-Ni2+ (Avanti Lipids, Cat# 790404P), and PEG-5000 PE (Avanti Lipids, Cat# 880230P) were first solubilized in chloroform to a stock concentration of 20 mg/mL, 10 mg/mL, and 1 mg/mL. Lipid mixture containing 98% POPC, 2% DGS-NTA-Ni2+, and 0.1% PEG-5000 PE was dried under a stream of nitrogen gas followed by vacuum pumping to evaporate chloroform thoroughly. The dried lipids were then resuspended in PBS to a final concentration of 0.5 mg/mL. Multilamellar vesicle solution was next solubilized by 1% w/v sodium cholate and loaded onto a desalting column. During the desalting process, sodium cholate was diluted to allow small unilamellar vesicles (SUVs) to form in the buffer containing 100 mM NaCl, 50 mM Tris-HCl (pH 7.8), and 1 mM TCEP (the 2D buffer).
3
Synthetic Lipid Vesicle Preparation
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Synthetic 1,2-dioleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl](nickel salt) (DGS-NTA-Ni), L-α-phosphatidylserine (Brain PS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)−2000] (ammonium salt) (DSPE-PEG 2000 Biotin), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)−5000] (ammonium salt) (PEG5000 PE) were purchased from Avanti Polar Lipids. Phospholipids for reconstitution assays (93% POPC, 5% PS, 2% DGS-NTA-Ni and 0.1% PEG 5000 PE) or (98% POPC, 1% DSPE-PEG2000 Biotin, 1% DGS-NTA-Ni, and 0.1% PEG5000-PE) were dried under a stream of Argon, desiccated over 3 hr, and resuspended in PBS (pH 7.3). To promote the formation of small unilamellar vesicles (SUVs), the lipid solution was repeatedly frozen in liquid N2 and thawed using a 37°C water bath until the solution cleared. Cleared SUV-containing solution was centrifuged at 33,500 g for 45 min at 4°C. Supernatant containing SUVs was collected and stored at 4°C covered with Argon.
4
Supported Lipid Bilayer Formation for Immune Cell Studies
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SLBs were formed on glass-bottomed 96-well plate (DOT Scientific Inc, #MGB096-1-2-LG-L). Briefly, the plate was cleaned with 2.5% Hellmanex (Sigma-Aldrich, #Z805939-1EA), etched with 5 N NaOH, and used for SLB formation as previously described (Ahrends et al., 2017 (link)). Briefly, small unilamellar vesicles (SUVs) derived from dried lipid film containing 95.5% POPC (Avanti Polar Lipids, #850457C), 2% biotin-DPPE (Avanti Polar Lipids, #870285P), 2% DGS-NTA-Ni (Avanti Polar Lipids, #790404C), and 0.1% PEG 5000-PE (Avanti Polar Lipids, #880230C) were added onto freshly treated plates to form SLBs. The SLBs were rinsed with wash buffer (1× PBS containing 0.1% BSA) and mixed with 1 μg/ml streptavidin, 0.1 nM His-tagged human PD-L1 ECD, and 3 nM His-tagged human ICAM-1 ECD at 37°C for 1 hr. Afterward, the SLBs were rinsed with wash buffer and further incubated with 5 μg/ml biotin anti-human-CD3ε at 37°C for 30 min, followed by three rinses with wash buffer and three rinses with imaging buffer (20 mM HEPES pH 7.5, 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 0.7 mM Na2HPO4, 6 mM D-glucose).
5
Fabrication of Small Unilamellar Vesicles
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The general protocol we follow to make small unilamellar vesicles (SUVs) and supported phospholipid bilayers is described in Su et al., 2017 (link). Synthetic 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt, DGS-NTA-Ni), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)–5000] (ammonium salt) (PEG5000 PE) were purchased from Avanti Polar Lipids as chloroform suspension. Using glass Hamilton syringes, lipids were mixed to make a chloroform suspension containing 98% POPC, 2% DGS-NTA-Ni and 0.1% PEG5000 PE. Chloroform was evaporated with gentle stream of Argon Gas, desiccated in a vacuum overnight, and resuspended in PBS (pH 7.3) with vortexing. To promote the formation of small unilamellar vesicles (SUVs), the lipid solution was repeatedly frozen in liquid N2 and thawed using a 37°C water bath until the solution cleared (~35 freeze-thaw cycles). SUV-containing solution was centrifuged at 33,500 g for 45 min at 4°C to remove large vesicles. Cleared supernatant containing SUVs was collected and stored at 4°C covered with Argon for up to two weeks.
6
Supported Lipid Bilayer Functionalization
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SLBs were formed on glass-bottomed 96-well plate (DOT Scientific Inc, #MGB096-1-2-LG-L). Briefly, the plate was cleaned with 2.5 % Hellmanex (Sigma-Aldrich, #Z805939-1EA), etched with 5N NaOH, and used for SLB formation as previously described 57 . Briefly, small unilamellar vesicles (SUVs) derived from dried lipid film containing 95.5% POPC (Avanti Polar Lipids, #850457C), 2% biotin-DPPE (Avanti Polar Lipids, #870285P), 2% DGS-NTA-Ni (Avanti Polar Lipids, #790404C) and 0.1% PEG 5000-PE (Avanti Polar Lipids, #880230C) were added onto freshly treated plates to form SLBs. The SLBs were rinsed with wash buffer (1x PBS containing 0.1% BSA) and mixed with 1 μg/mL streptavidin, 0.1 nM His-tagged human PD-L1 ectodomain, and 3 nM His-tagged human ICAM-1 ectodomain at 37 °C for 1 h. Afterward, the SLBs were rinsed with wash buffer and further incubated with 5 μg/mL biotin anti-human-CD3ε at 37 °C for 30 min, followed by three rinses with wash buffer and three rinses with imaging buffer (20 mM HEPES pH 7.5, 137 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 2 mM MgCl 2 , 0.7 mM Na 2 HPO 4 , 6 mM D-Glucose).
7
Supported Lipid Bilayer Formation from SUVs
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Small unilamellar vesicle (SUV) preparation: Phospholipids containing 98% POPC (Avantilipids), 2% DGS-NTA-Ni (Avantilipids) and 0.1% PEG 5000 PE (Avantilipids) were dried under a stream of nitrogen and resuspended by PBS to a final concentration of 0.5 mg/ml. The lipid solution was repeatedly frozen and thawed using a combination of liquid N 2 and 37 C water bath until the solution turned clear. Then the solution was subjected to a centrifugation at 33,500 g for 45 min at 4 C. Supernatant containing SUVs was collected. Chambered cover glass wash and lipid coating Chambered cover glass (Lab-tek) was initially washed with Hellmanex II (He ¨lma Analytics) overnight, thoroughly rinsed with MilliQ H 2 O. The cover glass was then washed with 5M NaOH for 1 hr at 50 C and thoroughly rinsed with MilliQ H 2 O, repeated for three times, and followed by equilibration with the Protein Buffer (50 mM Tris, pH 8.2, 100 mM NaCl, 1 mM TCEP). Typically, 150 mL SUVs were added to a cleaned chamber and incubated for 1 hr at 42 C, allowing SUVs to fully collapse on glass and fuse to form supported lipid bilayers (SLBs). SLBs were washed with the Protein Buffer for three times (6-folds dilution per time, 216-folds dilution in total) to remove extra SUVs. Then it was blocked with the Cluster Buffer (the Protein Buffer supplied with 1 mg/ml BSA) for 30 mins at room temperature.
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