μ slide v10.5 chip

Bilayers were prepared by bursting liposomes on the glass surface using a glass-bottomed μ-Slide V1^0.5 chip from Ibidi. Also, 2.5 µL MgCl₂ at 500 mM was added into 122.5 µL buffer containing 50 mM HEPES (pH 7.4), 140 mM KCl, and 10% glycerol. Then, 125 µL extruded bilayer liposomes were added. Next, 60 µL MgCl₂-liposome solution was loaded into the channel of the ibidi chip and incubated for 40 min at room temperature. The channel was washed with the same buffer supplemented with 6 mM EDTA and then with buffer supplemented with 1 mM DTT. Depending on the purpose of experiments, MgCl₂ or CaCl₂ may be added in the buffer to the desired concentration. Then, 60 µL of 10 nM Munc13-1-Halo-Alexa488 were loaded into the channel and incubate with the bilayer for 60 min at room temperature. The channel was washed with the buffer supplemented with 1 mM DTT. The vesicle liposomes were diluted 30 times. Subsequently, 60 µL diluted vesicle liposomes were loaded into the channel and incubated for 5 min at room temperature. The channel was washed with buffer supplemented with 1 mM DTT.
The Ibidi chip was then mounted to the stage of a Nikon TIRF microscope. Bilayers, Munc13 particles on bilayers, and vesicles attached to bilayers were respectively imaged at room temperature with the TIRF microscope using the corresponding laser.