RSC Advances
Page 6 of 8
ARTICLE
DOI: 10.1039/C6RA15938A
fluorescence intensity of QLBA in the absence of Cu2+, at a certain 2 (a) K. Y. Liu, H. M. Shang, F. F. Meng, Y. Liu, W. Y. Lin,
concentration of Cu2+ ion, the minimum fluorescence intensity of
[QLBAꢀCu2+] in the linear range, [M] is the Cu2+ concentration, n is
the binding stoichiometry.
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F − F
F − Fmin
0
log[
] = n log[M] + log Ka
Eq. 1
Cell cytotoxicity assay
To test the cytotoxicity of QLBA, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT) assay was performed. After
treatment with QLBA (2.0, 5.0, 10, 20 and 50 ꢁM) for 24 h, 10 ꢁL
1
of a MTT solution (5 mg mL− PBS) was added into each well of a
o
96ꢀwell culture plate and incubated continuously at 37 C for 4 h.
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Then all media were removed from wells and 100
ꢁL dimethyl
sulfoxide was added into each well. The optical density was
measured at 570 nm (OD570) wavelength with a Microplate Reader
(TECAN Infinite M1000PRO). The cell viability was expressed as
the optical density (OD) ratio of the treatment to control.
Cell viability and confocal imaging
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HeLa cells were cultured in RPMIꢀ1640 medium with 10% Fetal
Bovine Serum (FBS), 10 IU mLꢀ1 of penicillin, and 10 ꢁg mLꢀ1 of
streptomycin under a humidified atmosphere of 5% CO2 in air. The
concentrations of counted cells were adjusted to 1 × 106 cells/mL for
confocal imaging in RPMIꢀ1640 medium. Cells were incubated with
20 ꢁM QLBA in the culture medium for 4 h at 37 oC and then
incubated with 0 and 20 ꢁM of Cu2+ for 0.5 h at 37 C. Then, the
o
cells were washed three times with PBS and incubated with 10 and
20 ꢁM of sulfide ion in PBS at 37 °C for 0.5 h in a CO2 incubator.
After that the cells were washed with PBS another three times prior
to fluorescence imaging. All of the specimens were photographed
using the Olympus FV1000ꢀIX81 confocal laser scanning
microscope. Fluorescence images were obtained by excitation with a
multiline Hg laser and analyzed using ImageJ software (Wayne
Rasband, National Institutes of Health, Bethesda, MD).
Acknowledgment
We thank the advice of Dr. Lele Zhang (the Analytical & Testing
Center of Graduate School at Shenzhen, Peking University). This
work was supported by the National Natural Science Foundation of
China (No.21302108), Shenzhen Municipal government SZSITIC
(No. CXB201104210014A, JCYJ20140509151735023), and
Shenzhen Reform Commission (Disciplinary Development Program
for Chemical Biology).
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Notes and references
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