Spark fluorescence microplate reader

A Laurdan generalized
polarization (GP) assay was performed as previously described.^{32 (link)}E. coli ATCC 25922 was cultured
in 3 mL of MH II broth while shaking at 180 rpm at 37 °C overnight.
0.5 mL of cell suspension was diluted 10 times with 0.1% glucose MH
II broth to make OD₆₀₀ ∼ 0.1. The bacterial cells
were grown to the mid log phase (OD₆₀₀ ∼ 0.5). Laurdan
dye (Invitrogen, D250) was dissolved in DMSO (1 mM as a stock solution).
50 μL of 1 mM Laurdan stock solution was added to a 5 mL suspension
of the mid log phase cells. The resulting suspension was mixed gently
and incubated for 10 min at RT in the dark. The dye-treated cells
were centrifuged to 6000 rpm at RT for 5 min. The pellet was washed
with 5 mL of 0.1% glucose PBS three times and re-suspended with 2.5
mL 0.1% glucose PBS. Each peptide was serially diluted in 0.1% glucose
PBS to make a 2× peptide solution. A mixture of 50 μL of
the E. coli suspension and 50 μL of 2×
peptide solution was added to a well of a black 96-well chimney plate
(Greiner) and incubated for 1 h at 37 °C. The fluorescence intensity
of each sample was measured at λ_ex = 350 ± 20
nm and λ_em = 440 ± 20 nm, 490 ± 20 nm with
a SPARK fluorescence microplate reader (Tecan Life Science, Switzerland).
The GP value of the Laurdanstained bacterial cells with or without
each peptide was determined using the following formula Independent triplicates were performed.

A 1-N-phenylnaphthylamine
(NPN) assay was carried out using a previously described method.^{31 (link)} Briefly, E. coli ATCC 25922
was cultured in 3 mL of LB broth while shaking at 180 rpm at 37 °C
overnight and diluted to OD₆₀₀ ∼ 0.1 in 5 mL of
LB broth. The bacterial cells were grown to the mid log phase (OD₆₀₀ ∼ 0.5) and centrifuged to 6000 rpm at RT for 10
min. The cells were re-suspended in a half volume of 5 mM HEPES buffer
(pH 7.2). NPN was dissolved in DMSO (50 mM as a stock solution). The
NPN stock solution was diluted to 40 μM using 5 mM HEPES buffer
(pH 7.2). Each peptide was serially diluted in 5 mM HEPES buffer (pH
7.2). 50 μL of 4× peptide solution and 50 μL of NPN
solution were added to a well in a black 96-well chimney plate (Greiner).
100 μL of E. coli suspension was transferred
into the mixture. After pipetting 10 times, the fluorescence intensity
of the resulting mixture was measured immediately (NPN at λ_ex = 355 ± 20 nm and λ_em = 405 ±
20 nm). The fluorescence intensity of each sample was calculated by
subtracting that of the blank. The fluorescence intensity of each
sample was measured using a SPARK fluorescence microplate reader (Tecan
Life Science, Switzerland). Independent triplicates were performed.

BODIPYTR cadaverine
(BC) was used to measure the relative binding efficiency of each peptide
to LPS utilizing a slight modification of the previously described
method.^{27 (link)} An LPSBC mixture was prepared
by mixing 50 μg/mL LPS from E. coli O128:B12
and 2.5 μM BC in 50 mM Tris buffer (pH 7.4). Each peptide was
serially diluted in 50 mM Tris buffer (pH 7.4). 50 μL of the
resulting mixture was transferred into a 96-well chimney black plate
(Greiner) and mixed by gentle pipetting. After incubation at 37 °C
for 1 h, the fluorescence intensity of each sample was measured at
λ_ex = 580 ± 20 nm and λ_em =
620 ± 20 nm with a SPARK fluorescence microplate reader (Tecan
Life Science, Switzerland). The BC occupancy factor was calculated
with the following formula
The maximum fluorescence intensity
was the fluorescence intensity at the maximum PMB concentration. The
minimum fluorescence intensity was the fluorescence intensity without
the peptide. Independent triplicates were performed.