1 2 dioleoyl sn glycero 3 phosphoethanolamine n cap biotinyl sodium salt

Measurements of binding kinetics of LFA1 and ICAM1 were performed as described in our previous studies.^{19 (link),22 (link)} Briefly, mouse ICAM1-GPI was purified, labeled with ATTO647N-NHS-ester, and incorporated into liposomes consisting of 0.4 mM 1,2-dioleoyl-sn-glycero-3-phosphocholine containing 0.1 mol% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (Avanti Polar Lipids). Liposomes harboring ICAM1-GPI were applied onto piranha-treated cover glass to generate supported planar lipid bilayers presenting ICAM1. Single-molecule binding events between LFA1 and ICAM1 on primary T cells (WT or Rasa3/Sipa1-KO) were recorded at 33-msec intervals on a total internal reflection microscope (TIRFM, Olympus) in the buffer containing 20 mM HEPES (pH 7.4), 140 mM NaCl, 2 mg/mL glucose, 0.5 mM CaCl₂, and 0.5 mM MgCl₂. When bound to LFA1, rapidly moving ICAM1 decelerated or arrested, exhibiting diffusion coefficients two orders of magnitude smaller than unbound ICAM1. Single-particle tracking of ICAM1 was performed using the G-count and G-track software (G-angstrom). Frequencies and bond lifetimes extracted from trajectories of bound ICAM1 were used for the calculation of dissociation rate constants.^{22 (link)}