Biocoat poly d lysin laminin

Melittin (0.1 μM,
monomer equivalents) or α-hemolysin (50 μg/mL, monomer
equivalents) were added to the cell culture media of SH-SY5Y cells
seeded on glass coverslips (Corning BioCoat Poly-d-Lysin/Laminin,
NY) for 5 or 1 min, respectively, in the absence or presence of 0.01–10
μM claramine. To detect intracellular ROS production, cells
were loaded with 10 μM 6-chloromethyl-2′,7′-dichlorodihydrofluorescein
diacetate (CM-H₂DCFDA, Life Technologies, CA) during the
aforementioned treatment. The resulting fluorescence was analyzed
by a Nikon C2 scanning laser confocal microscopy system (Nikon Instruments,
NY). A series of 1.0 μm thick optical sections (1024 ×
1024 or 2048 × 2048) were taken through the cells using a Nikon
Eclipse Ti inverted microscope (Nikon Instruments) equipped with a
60× oil immersion objective (Nikon Instruments) and then projected
as a single composite image by superimposition. The confocal microscope
was set at optimal acquisition conditions, e.g., pinhole diameters,
detector gain, and laser powers. Settings were maintained constant
for all image acquisitions. For Figures 1C and 3B, the same
images are shown with enhanced brightness and contrast such that all
cells can be visualized (Figure S9), including
those with low fluorescence signals.

To label
melittin, 300 μM Alexa Fluor 488 N-hydroxysuccinimide
(NHS) ester (succinimidyl ester, Invitrogen, ThermoFisher Scientific,
CA) was incubated with gentle shaking for 2 h with 900 μM melittin
in 0.1 mM sodium bicarbonate buffer (pH 8.0, Sigma-Aldrich, MO). SH-SY5Y
cells were seeded on glass coverslips (Corning BioCoat Poly-d-Lysin/Laminin, NY) and treated for 5 min with 0.2 μM labeled
melittin in the absence or presence of 0.1, 1.0, and 10 μM claramine.
After incubation, the cells were washed with phosphate-buffered saline
(PBS) and counterstained with 5 μg/mL Alexa Fluor 633-conjugated
wheat germ agglutinin (Life Technologies, CA).^{13 (link)} After washing with PBS, cells were fixed in 2% paraformaldehyde.
Fluorescence emission was detected after double excitation at 488
and 633 nm by the above-described scanning confocal microscopy system
using a 60× oil immersion objective (Nikon Instruments). A series
of 1.0 μm thick optical sections (1024 × 1024) were acquired,
and all sections were projected as a single composite image by superimposition.
ImageJ (NIH, Bethesda, MD) was used to calculate the percentage of
colocalization between cell membranes and melittin.