The preparation of planar lipid bilayers is detailed elsewhere (Grakoui et al., 1999 (link); Carrasco et al., 2004 (link)). Bilayers were prepared that contained biotin lipids to which biotinylated ICAM-1 and antigens were attached through streptavidin. In brief, PC10-BSA, NIP16-BSA, and the mouse ICAM-1/huFc chimera protein (R&D Systems) were biotinylated with EZ-link sulfo-NHS-LC-biotin (Thermo Fisher Scientific). An aliquot of each was labeled with sulfo-NHS–functionalized fluorophores (Invitrogen) to allow monitoring of the mobility of the lipid-anchored proteins in the lipid bilayers. Biotin-labeled small unilamellar lipid vesicles were prepared by mixing a 100:1 molar ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-cap-biotin (Avanti Polar Lipids, Inc.). The lipid mixture was sonicated and resuspended in PBS at a lipid concentration of 5 mM. Aggregated lipid vesicles were cleared by ultracentrifugation and filtering.
Bilayers were formed in Lab-Tek chambers (Thermo Fisher Scientific) in which the coverglasses were replaced with nanostrip-washed coverslips. The coverslips were incubated with 0.1 mM biotin-labeled small unilamellar lipid vesicles in PBS for 10 min. After washing with 20 ml PBS, the bilayer was incubated with 2.5 μg/ml streptavidin for 10 min, and excess streptavidin was removed by washing with 20 ml PBS. The bilayers were incubated for 20 min with 0.5 μg/ml of biotinylated mouse ICAM-1, and excess ICAM-1 was removed by washing. The streptavidin- and ICAM-1–containing planar lipid bilayers were incubated with 0.75 μg/ml of biotinylated PC-BSA or NIP-BSA. The unbound excess of antigen was removed by washing with 20 ml PBS. The mobility of ICAM-1 and antigens in the lipid bilayers was confirmed by analyses of the proteins labeled with fluorescent dyes.
Alternatively, NIP- and ICAM-1–containing planar lipid bilayers were prepared by fusing small unilamellar lipid vesicles with a clean glass coverslip surface as described previously (Brian and McConnell, 1984 (link)) using 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl] in nickel salt (Avanti Polar Lipids, Inc.) at a 10:1 ratio. Small unilamellar vesicles were obtained by sonication and clarified by ultracentrifugation and filtering. Glass coverslips were cleaned in Nanostrip (Cyantek), washed, and dried. Lipid bilayers were prepared from a 0.1-mM lipid solution on the coverslips attached to the bottom of Lab-Tek imaging chambers. After excess lipids were washed away, histidine-tagged antigens and ICAM-1 were bound. Before imaging, chambers were washed with HBSS supplemented with 1% FCS. NIP14-BSA was prepared as described previously (Tolar et al., 2005 (link)) and conjugated to a cystein-containing peptide terminated with a 12-histidine tag (ASTGTASACTSGASSTGSH12) using SMCC (Thermo Fisher Scientific) according to the manufacturer's protocols. Recombinant ICAM-1 tagged with a 12-histidine tag was a gift from J. Huppa (Stanford University, Palo Alto, CA). Conjugation of NIP14-BSA to succinimidyl AlexaFluor647 and ICAM-1-H12 to AlexaFluor488 (both obtained from Invitrogen) was performed according to the manufacturer's protocols.
Bilayers were formed in Lab-Tek chambers (Thermo Fisher Scientific) in which the coverglasses were replaced with nanostrip-washed coverslips. The coverslips were incubated with 0.1 mM biotin-labeled small unilamellar lipid vesicles in PBS for 10 min. After washing with 20 ml PBS, the bilayer was incubated with 2.5 μg/ml streptavidin for 10 min, and excess streptavidin was removed by washing with 20 ml PBS. The bilayers were incubated for 20 min with 0.5 μg/ml of biotinylated mouse ICAM-1, and excess ICAM-1 was removed by washing. The streptavidin- and ICAM-1–containing planar lipid bilayers were incubated with 0.75 μg/ml of biotinylated PC-BSA or NIP-BSA. The unbound excess of antigen was removed by washing with 20 ml PBS. The mobility of ICAM-1 and antigens in the lipid bilayers was confirmed by analyses of the proteins labeled with fluorescent dyes.
Alternatively, NIP- and ICAM-1–containing planar lipid bilayers were prepared by fusing small unilamellar lipid vesicles with a clean glass coverslip surface as described previously (Brian and McConnell, 1984 (link)) using 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl] in nickel salt (Avanti Polar Lipids, Inc.) at a 10:1 ratio. Small unilamellar vesicles were obtained by sonication and clarified by ultracentrifugation and filtering. Glass coverslips were cleaned in Nanostrip (Cyantek), washed, and dried. Lipid bilayers were prepared from a 0.1-mM lipid solution on the coverslips attached to the bottom of Lab-Tek imaging chambers. After excess lipids were washed away, histidine-tagged antigens and ICAM-1 were bound. Before imaging, chambers were washed with HBSS supplemented with 1% FCS. NIP14-BSA was prepared as described previously (Tolar et al., 2005 (link)) and conjugated to a cystein-containing peptide terminated with a 12-histidine tag (ASTGTASACTSGASSTGSH12) using SMCC (Thermo Fisher Scientific) according to the manufacturer's protocols. Recombinant ICAM-1 tagged with a 12-histidine tag was a gift from J. Huppa (Stanford University, Palo Alto, CA). Conjugation of NIP14-BSA to succinimidyl AlexaFluor647 and ICAM-1-H12 to AlexaFluor488 (both obtained from Invitrogen) was performed according to the manufacturer's protocols.
