Ability To Interact with LPS-BODIPY

Binding and dissociation of LPSs were performed using a previously published protocol.⁵¹ Briefly, a stock solution of 100 μg/mL of E. coli BODIPY-LPS [BODIPYFL (503/513), Molecular Probes, Life Technologies, USA] was diluted to 10 μg/mL in 1× phosphate-buffered saline (PBS; pH 7.4) and sonicated for 2 min every time prior to use. Two milliliters of this sonicated solution of BODIPY-LPS was transferred to a quartz cuvette at a concentration of 500 ng/mL. Subsequently, the respective concentrations of NCK-10 in PBS were added to it, and the fluorescence was measured using a λS55 fluorescence spectrophotometer (PerkinElmer). The parameters used were as follows: excitation wavelength: 485 nm; emission wavelengths: 500–700 nm; excitation slit width = 5 nm; emission slit width = 15 nm; and temperature: 25 °C. Three independent experiments were performed, and the data furnished are a representative image of one experiment.