Dopamine-modified cationic conjugated polymer as a new platform for ph sensing and autophagy imaging

Article abstract of DOI:10.1002/adfm.201202132

A dopamine-modified conjugated polymer PFPDA is synthesized and characterized. At low pH, dopamine exists in its hydroquinone form and lacks the ability to quench fluorescence. At high pH, the proportion of the quinone form of dopamine increases due to its autooxidation, and efficient intramolecular electron transfer from the polymer main chain to quinone occurs, resulting in the quenching of the fluorescence of PFPDA. Thus, PFPDA exhibits a fluorescence turn-on response at low pH. PFPDA possesses excellent photostability and exhibits no cytotoxicity, which makes it a good fluorescent material for pH sensing and cell imaging. A light-induced hydroxyl anion emitter, MGCB, is also used to change the pH of the solution and thus regulate the fluorescence of PFPDA via remote control under light irradiation. Because the cytoplasm becomes acidic when cell autophagy occurs, PFPDA can also be used for autophagy imaging of HeLa cells with good selectivity. A dopamine-modified conjugated Polymer, PFPDA is synthesized for pH sensing. At low pH, dopamine exists in its hydroquinone form and lacks the ability to quench fluorescence. At high pH, the quinone from dopamine autooxidation quenches the fluorescence of PFPDA through efficient intramolecular electron transfer. PFPDA exhibits a fluorescence turn-on response at low pH, and can be used for autophagy imaging of HeLa cells with good selectivity. Copyright

Full text of DOI:10.1002/adfm.201202132

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Dopamine-Modified Cationic Conjugated Polymer as a New
Platform for pH Sensing and Autophagy Imaging
Quanshan Wen, Libing Liu,* Qiong Yang, Fengting Lv, and Shu Wang*
inherent cytotoxicity limits their further
application in cell biology.[10,11] Therefore,
A dopamine-modified conjugated polymer PFPDA is synthesized and char-
it is of great significance to develop other
kinds of fluorescence systems for pH
sensing.
acterized. At low pH, dopamine exists in its hydroquinone form and lacks
the ability to quench fluorescence. At high pH, the proportion of the quinone
form of dopamine increases due to its autooxidation, and efficient intramo-
Conjugated polymers (CPs), coordi-
lecular electron transfer from the polymer main chain to quinone occurs,
resulting in the quenching of the fluorescence of PFPDA. Thus, PFPDA
exhibits a fluorescence “turn-on” response at low pH. PFPDA possesses
excellent photostability and exhibits no cytotoxicity, which makes it a good
fluorescent material for pH sensing and cell imaging. A light-induced hydroxyl
anion emitter, MGCB, is also used to change the pH of the solution and thus
regulate the fluorescence of PFPDA via remote control under light irradiation.
Because the cytoplasm becomes acidic when cell autophagy occurs, PFPDA
can also be used for autophagy imaging of HeLa cells with good selectivity.
nating the action of many repeated units
and possessing strong light-harvesting
and significant optical signal amplification
properties, serve as a platform for highly
sensitive biological and chemical detec-
tions.[12,17] Several conjugated polymers
containing various cationic or anionic
moieties have been reported to establish
fluorescence pH-sensing systems on the
basis of the different aggregation forms of
CPs in different pH environments.[1,18–21]
However, for most of these systems, the
fluorescence is “turned-off” at low pH
(acidic condition). In the meantime, the pH-response range
of these CPs is often not located around physiological pH due
to the intrinsic pKa values of the cationic or anionic moieties.
Thus, application of these CPs in cell biology is limited. In this
paper we report a “turn-on” pH sensing system at low pH by
linking a dopamine moiety to a conjugated polymer to afford
dopamine-modified polyfluorene derivative (PFPDA). This
system is not based on an aggregation effect but the redox prop-
erty of dopamine. The oxidized dopamine form (quinone) dom-
inates in aqueous media with high pH and thus the quenching
effect toward the conjugated polymer can be regulated by the
pH change.[22] The pH-depentent redox property of dopamine
and the signal amplification characteristics of CPs are combined
to realize rapid, sensitive, and acid-responsive pH sensing. An
organic dye, malachite green carbinol base, is reported to have
the ability of releasing hydroxyl anions and thus altering the
pH of solution via ultraviolet irradiation,[23,24] therefore it can be
utilized in regulation of the fluorescence of PFPDA via remote
control under light irradiation. Finally, this pH sensing system
is also able to be applied in the autophagy imaging of HeLa
cells (human cervical carcinoma cell line) because a portion of
cytoplasm becomes acidic when autophagy occurs.[25,26]
1. Introduction
In recent decades, pH sensing in aqueous media has attracted a
great deal of interest in many fields such as analytical chemistry,
cellular biology, medicine and environmental protection.[1,2]
The acid-response pH sensing of cellular environment is quite
crucial for many reasons. For example, the decrease of intracel-
lular pH can cause apoptosis,[3] and acidic organelles such as
lysosomes and endosomes also functionalize in endocytic and
digestive processes.[4] Fluorescence technology is one of the
important methods to achieve rapid and sensitive pH sensing
due to the advantage of high sensitivity and low background
noise, thus it is widely applied in pH sensing.[5] Fluorescence
pH sensing can be achieved by the use of many kinds of sub-
stances, such as quantum dots (QDs), fluorescent proteins and
other small organic dyes, via intramolecular or intermolecular
charge/energy transfer mechanisms.[6–8] However, each kind of
method has its own limitations despite of its specific advantage.
For example, small organic dyes usually suffer from low water
solubility and comparatively poor photostability, which limit
their use in biological research.[9] QDs possess advantages of
narrow emission, and high brightness and photostability, but
Q. Wen, Dr. L. Liu, Dr. Q. Yang, Dr. F. Lv, Prof. S. Wang
Beijing National Laboratory for Molecular Science
Key Laboratory of Organic Solids
2. Results and Discussion
Institute of Chemistry
Chinese Academy of Sciences
Beijing 100190, China
E-mail: liulibing@iccas.ac.cn; wangshu@iccas.ac.cn
Our new CP-based pH assay is illustrated in Scheme 1. The
dopamine is covalently linked to the side chain of a water-soluble
conjugated polymer (PFPDA). Dopamine can exhibit different
redox states and change between hydroquinone (reduced state)
and quinone (oxidized state) reversibly in aqueous media with a
DOI: 10.1002/adfm.201202132
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continuous irradiation by a mercury lamp (100 W) at 380 nm
for 60 s. Thus, PFPDA exhibits excellent photostability, which
is significant for further sensing and imaging investigation.
The quantitative cytotoxicity of PFPDA was measured using an
MTT method. As shown in Figure 1c, the cell viability of HeLa
cells did not decrease after 24 h incubation with varying PFPDA
concentration (0–100 μM). The low toxicity of PFPDA is critical
for its application in cell imaging.
The pH response of PFPDA was investigated in PBS with
different pH. As shown in Figure 2a, the fluorescence intensity
of PFPDA increased as the pH changed from high to low. The
maximum emission intensity at 455 nm decreases linearly with
increasing pH value in the pH range of 5.0–9.0 (Figure 2b).
For this system, there is a good response around neutral pH
(6.0–8.0), facilitating utilization in a biological environment. At
low pH, dopamine exists as its hydroquinone form and lacks
the ability to quench the fluorescence of PFPDA. At higher pH,
the proportion of quinone of dopamine increases due to its
autooxidation. This results in electron transfer from PFPDA to
Scheme 1. Schematic representation of pH sensing on the basis of
PFPDA.
change in pH.[22] At low pH, dopamine exists
as its hydroquinone form and lacks the ability
to quench fluorescence. The electron transfer
(ET) from the polymer main chain (donor) to
hydroquinone (acceptor) is absent and strong
emission is observed upon excitation of the
polymer. As pH is increased, the proportion
of quinone form of dopamine increases due
to its autooxidation. In this case, efficient
intramolecular ET from polymer main chain
to quinone occurs and the fluorescence of
PFPDA is quenched. Thus, a different fluo-
rescent response of the PFPDA is expected as
the environmental pH changes.
The synthetic routes for monomers and the
polymer PFPDA are outlined in Scheme 2.
3-(2-((6-bromohexyl)oxy)ethyl)thiophene (1)
was treated with N-iodosuccinimide (NIS) to
afford compound 2. Via a multistep reaction,
the compound 1 was converted to compound
3 followed by conjugation with dopamine to
give monomer 4. Coupling monomers 4, 5,
and 6 via a Suzuki reaction in the presence
of catalyst Pd(dppf)Cl2 gave polymer PFPDA
with 47% yield. The ratio of the two copoly-
1
merized units was calculated from H NMR
to be 5/1. The PFPDA was dissolved in water
containing 5% DMSO (v/v) at room tempera-
ture to be used for further experiments.
The photophysical properties of PFPDA
were investigated in phosphate buffered
saline (PBS). As shown in Figure 1a, the
UV-vis absorption spectrum of PFPDA shows
a maximum peak at 380 nm, while the emis-
sion spectrum exhibits a maximum peak at
455 nm and a shoulder peak at 425 nm upon
excitation at 390 nm. We studied the fluores-
cence intensity of PFPDA under light irra-
diation. Figure 1b shows that PFPDA main-
tained its original fluorescence intensity upon
Scheme 2. The synthetic route of PFPDA.
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Figure 1. a) Normalized UV-vis absorption and emission spectra of PFPDA in PBS (pH = 7.0). The excitation wavelength is 390 nm. b) Photostability
of PFPDA upon irradiation at 380 nm under a mercury lamp (100 W). c) Cell viability of HeLa cells incubated with various concentrations of PFPDA.
Standard deviations calculated from six replicate samples.
Figure 2. a) Fluorescence spectra of PFPDA in PBS at different pH values (5.0, 6.0, 7.0, 8.0, and 9.0). b) The maximum fluorescence intensity of PFPDA
at 455 nm as a function of pH value; [PFPDA] = 1 μM in RUs. The excitation wavelength is 390 nm. c) Absorption spectra of PFPDA in PBS at pH 5.0
and 9.0. Measurements were performed in phosphate buffered saline solution; [PFPDA] = 10 μM. d) Absorption spectra of PFPDA at pH 9.0 between
420 and 480 nm with subtraction of the residual absorbance of PFPDA at pH 5.0.
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quinone and the fluorescence of PFPDA is quenched. The for-
mation of quinone from dopamine was verified by UV-vis spec-
troscopy. Figure 2c compares the absorption spectra of PFPDA
in PBS at pH 5.0 and 9.0; an intensity increase at 440 nm is
observed for pH 9.0 (Figure 2d), which provides the evidence
of the autooxidation of dopamine from its hydroquinone to qui-
none form.[27] The fluorescence quantum yields of PFPDA in
aqueous solution were also measured with the value of 2% at
pH 9.0 and 11% at pH 5.0.
Organic dye, malachite green carbinolbase (MGCB), was
utilized to achieve the regulation of fluorescence of PFPDA.
It is reported that MGCB can release hydroxide anions in the
presence of UV light and recombine with the OH− after the
UV light is stopped,[23–24] thus the solvent pH can be changed
by the hydroxide anions released by MGCB. In order to
improve the solubility of MGCB in aqueous media, the sur-
factant cetyltrimethylammonium bromide (CTAB) was used
in our experiments. The working system was established by
adding MGCB and PFPDA to the aqueous solution containing
CTAB (50 mM) and NaCl (200 mM). The UV-vis spectra of the
solution were recorded under 254 nm UV light. As shown in
Figure 3a, the absorbance at 621 nm increases as the solution
was irradiated under the 254 nm UV light, elucidating the gen-
eration of hydroxyl anion. It is noted that the peak at 621 nm
reaches a maximum after irradiation for about 15 min, which
shows that MGCB is completely converted to the malachite
green cation. In this case, the solution exhibited a deep green
color due to the formation of the malachite green cation. The
fluorescence spectra of PFPDA in this working system were
then measured upon irradiation under 254 nm UV light. The
fluorescence spectra were recorded every 3 min for 15 min.
As shown in Figure 3b, the maximum emission intensity of
PFPDA at 425 nm decreased by a half after 15 min irradiation.
The control experiment showed that the maximum emission
intensity of PFPDA did not change in the absence of MGCB.
These experiments showed that fluorescence regulation of
PFPDA was achieved by the MGCB-induced pH change via
UV irradiation. It is noted that the fluorescence of PFPDA can
recover to its original intensity after 30 min with the UV light
switched off (Figure 3c). The cycling of the fluorescence inten-
sity of PFPDA at 425 nm in the presence of MGCB under exci-
tation at 390 nm was realized by turning the UV light on and
off alternately (each cycle consisted of 20 min with light and
40 min without light). From the six cycles shown in Figure 3d,
we can see that the intensity of each cycle only slightly
decreased. In general, this system showed comparatively excel-
lent cycling properties.
Finally, PFPDA was applied to autophagy imaging of HeLa
cells. The HeLa cells were treated with rapamycin (25 μM) for
8 h to induce autophagy followed by staining with PFPDA.
Figure 3. a) UV-vis absorption spectra of PFPDA and MGCB under irradiation at 254 nm UV light for 0–20 min; [PFPDA] = 2.5 μM, [MGCB] = 100 μM.
b) Maximum fluorescence intensity of PFPDA at 425 nm with and without the addition of MGCB under irradiation at 254 nm for 15 min. The excitation
wavelength is 390 nm; [PFPDA] = 1 μM, [MGCB] = 100 μM. c) Fluorescence spectra of PFPDA in the presence of MGCB after the sample was irradiated
at 254 nm for 15 min and kept in the dark for another 15 min and 30 min. The excitation wavelength is 390 nm; [PFPDA] = 2 μM, [MGCB] = 100 μM.
d) Cycling of maximum fluorescence intensity of PFPDA at 425 nm in the presence of MGCB with the UV light on and off. The excitation wavelength
is 390 nm; [PFPDA] = 2 μM, [MGCB] = 100 μM.
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4. Experimental Section
Materials: Solvents were obtained from Beijing Chemical Co. and
used without further purification. THF was dried under sodium. DMF
was distilled under P2O5. 2,7-Dibromofluorene, Pd(dppf)Cl2 (98%)
was obtained from Pacific ChemSource, Inc. (China), malachite green
carbinol base and rapamycin were purchased from Sigma-Aldrich.
Dopamine hydrochloride and cetyltrimethylammonium bromide (CTAB)
were purchased from Alfa-Aesar.
Instruments: 1H NMR and 13C NMR spectra were recorded on a Bruker
AV400 instrument. Mass spectra were measured on a SHIMADZU
LCMS-2010 spectrometer for ESI, a Bruker Biflex spectrometer for
MALDI-TOF, and a Waters GCT spectrometer for high resolution mass
spectra (HRMS). Elemental analysis was performed on a Flash EA1112
instrument. UV-vis absorption spectra were performed using a JASCO
V-550 spectrophotometer. Florescence spectra were recorded on a
Hitachi F-4500 fluorometer equipped with a xenon lamp as excitation
source at room temperature, and the PMT voltage was 700 V. The pH
value was measured on a Mettler Toledo FE20 pH indicator, calibrated
with standard buffers of pH = 4.00, 7.00, and 10.00 at room temperature.
Phase contrast bright-field and fluorescence images were taken with
fluorescence microscopy (Olympus 1 × 71) with a mercury lamp
(100 W) as the excitation source. MGCB was irradiated in a WD-9403F
UV viewing cabinet.
Figure 4. Phase contrast image (a) and fluorescence image (b) of
rapamycin-treated HeLa cells stained with PFPDA. Phase contrast image
(c) and fluorescence image (d) of HeLa cells stained with PFPDA without
treatment with rapamycin. [PFPDA] = 10 μM, [rapamycin] = 25 μM.
Synthesis of 3-(2-((6-Bromohexyl)oxy)ethyl)-2,5-diiodothiophene (2) : To
a stirred solution of 3-(2-((6-bromohexyl)oxy)ethyl)thiophene (1) (1.0 g,
3.4 mM) in a 1:1 (v/v) solvent mixture of dichloromethane and acetic
acid (40 mL) was slowly added N-iodosuccinimide (1.93 g, 8.6 mM) at
0°C in the dark. The mixture was stirred at room temperature overnight
and then was washed with 10% sodium thiosulfate, saturated aqueous
NaHCO3 and brine. The organic phase was dried over Mg2SO4, filtered
and the solvent was removed under vacuum. Column purification was
performed on silica gel with petroleum ether/ethyl acetate (40:1) as the
eluent solvents to afford the pure compound as a white powder (1.55 g,
94%). 1H NMR (400 MHz, CDCl3) δ 6.98 (s, 1H), 3.54 (t, 2H), 3.42 (dd,
4H), 2.80 (t, 2H), 1.91–1.84 (m, 2H), 1.61–1.54 (m, 2H), 1.49–1.34 (m,
4H). 13C NMR (100 MHz, CDCl3) δ 146.13, 138.33, 77.99, 75.83, 70.77,
69.71, 33.93, 32.76, 32.44, 29.48, 27.96, 25.40. MS (MALDI-TOF): m/z
540.6 (M–H+); C12H17BrI2OS: calculated for C 26.54, H, 3.16; found C
26.93, H 3.26.
Synthesis of 6-(2-(2,5-Diiodothiophen-3-yl)ethoxy)hexanoic acid (3) : To a
solution of compound 2 (1.0 g, 1.8 mM) in 15mL DMF was added KOAc
(1.8 g, 18 mM). The mixture was vigorously stirred at 60 °C for 2 h under
nitrogen atmosphere. After cooling to room temperature the solvent was
removed under reduced pressure. The residue was treated with KOH
(1.24 g, 22 mM) in methanol (15 mL) at 60 °C for 2 h. The mixture was
concentrated and extracted with dichloromethane, followed by washing
by water and drying over Mg2SO4. After evaporation of the solvent, the
compound acquired was dissolved in acetone and the solution was
cooled to 0 °C. Jones’ reagent was added dropwise until the orange color
persisted and the mixture was stirred at room temperature overnight.
After isopropanol was added dropwise to quench the reaction, the
solvent was removed under vacuum. The solid was dissolved in 1 M HCl
and extracted with CH2Cl2. The combined organic layers were extracted
with 1 M NaOH, and the aqueous layer was then acidified followed by
extracting with CH2Cl2. The combined organic layers were dried over
anhydrous Mg2SO4, filtered and concentrated under reduced pressure
to give a yellow solid (628 mg, 69%). 1H NMR (400 MHz, CDCl3) δ
11.29 (s, 1H), 6.97 (s, 1H), 3.54 (t, 2H), 3.43 (t, 2H), 2.79 (t, 2H), 2.37
(t, 2H), 1.69–1.55 (m, 4H), 1.43–1.36 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 179.69, 146.04, 138.29, 78.00, 75.85, 70.65, 69.71, 33.96, 32.38,
29.27, 25.69, 24.45. HRMS (ESI) calculated for C12H16I2O3S [(M+H)+]:
494.8983, found: 494.8963.
The pH of lysosomal internal environment is around 5.0 and
thus is acidic compared with the slightly basic cytosol with pH
7.2. In the process of autophagy, a portion of the cytoplasm is
sequestered in autophagosome and eventually fused with lys-
osomes, leading to a pH decrease of the cytoplasm and thus
the increase of fluorescence intensity from the PFPDA in the
cells. As shown in Figure 4a,b, the HeLa cells stained with
PFPDA exhibit brighter emission intensity with the addition
of rapamycin compared with the control experiment (without
treatment with rapamycin) (Figure 4c,d). The brightness
intensity of the fluorescence of PFPDA in HeLa cells treated
with rapamycin is five times higher than that of cells without
treatment with rapamycin. It is noted that no difference was
observed when we used the commercially available dye (mono-
dansylcadaverine (MDC)) to image autophagy of HeLa cells
with and without treatment of rapamycin. Thus, our new pH
sensing system can be applied in the autophagy imaging of
cells with good selectivity.
3. Conclusions
A dopamine-modified conjugated polymer PFPDA was syn-
thesized and characterized, exhibiting a fluorescence “turn-
on” response at low pH values. PFPDA possesses excellent
photostability and exhibits no cytotoxicity, which makes it a
good fluorescent material for pH sensing and cell imaging.
We utilized the redox properties of dopamine to realize
a reversible pH response of the fluorescence intensity of
PFPDA. A light-induced hydroxyl anion emitter MGCB was
also used to change the pH of the solution and thus regulate
the fluorescence of PFPDA via remote control under light.
PFPDA was also used for turn-on autophagy imaging of HeLa
cells because a portion of the cytoplasm becomes acidic when
autophagy occurs.
Synthesis of N-(3,4-Dihydroxyphenethyl)-6-(2-(2,5-diiodothiophen-3-yl)
ethoxy)hexanamide (4) : To a solution of compound 3 (200 mg, 0.4 mM)
in DMF (6 mL) at 0 °C was sequentially added NEt3 (340 μL, 2.4 mM),
HOBt (110 mg, 0.8 mM), EDCI (170 mg, 0.9 mM), and dopamine
hydrochloride (230 mg, 1.2 mM). The reaction mixture stirred at 0 °C
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for 1 h and at room temperature overnight. After DMF was removed
under reduced pressure, 10 mL water was added and the aqueous layer
was extracted with chloroform. The collected organic layer was washed
with 1 M HCl. The organic layer was dried over Mg2SO4, filtered and
concentrated under vacuum. Column purification was performed on
silica gel with petroleum ether/ethyl acetate (1:2) as eluent solvents to
afford a colorless liquid (167 mg, 65.5%). 1H NMR (400 MHz, CDCl3)
δ 7.88 (s, 1H), 6.94 (s, 1H), 6.81 (d, 1H), 6.69 (s, 1H), 6.57 (d, 1H),
6.19 (s, 1H), 5.66 (s, 1H), 3.57 (t, 2H), 3.50–3.44 (m, 4H), 2.80 (t,
2H), 2.69 (t, 2H), 2.16 (t, 2H), 1.63–1.51 (m, 4H), 1.37–1.29 (m, 2H).
13C NMR (100 MHz, CDCl3) δ 174.25, 145.80, 144.32, 143.12, 138.20,
130.44, 120.36, 115.64, 115.37, 78.17, 75.97, 70.64, 69.59, 41.02, 36.59,
34.77, 32.22, 29.10, 25.73, 25.43. HRMS (ESI) calculated for C12H16I2O3S
[M+H]: 629.9669, found: 629.9653.
Synthesis of PFPDA: To a solution of monomer 5 (154 mg, 0.2 mM),
monomer 4 (31 mg, 0.05 mM) and monomer 6 (62 mg, 0.25 mM)
in 8 mL THF and 2 mL DMF was added 2 mL of aqueous potassium
carbonate (2.0 M). The resulting mixture was degassed and then
Pd(dppf)Cl2 (15 mg, dppf = diphenylphosphinoferrene) was added
under a nitrogen steam. The mixture was vigorously stirred at 80 °C for
2 days and then cooled down to room temperature. The solvents were
removed under vacuum and the solid was dissolved in methanol and
precipitated by acetone. The precipitate was collected and dialyzed in
water using a dialysis membrane with a cut-off of 3500 g mol−1 for two
days to yield a yellow solid (115 mg, 47%). 1H NMR (400 MHz, DMSO)
δ 8.58, 7.98–7.80, 7.57–7.36, 7.16, 6.62, 3.34, 3.17, 2.95, 2.08, 1.46, 1.14–
1.09, 0.66.
Assay for Photostability of PFPDA: The solution containing PFPDA was
dropped on a glass plate and covered with a coverslip. The sample was
continuously irradiated by a mercury lamp (100 W) with a 380/30 nm
excitation filter. Fluorescence emission intensities of the sample was
recorded with fluorescence microscopy (Olympus 1 × 71).
In Vitro Cell Viability Assay: HeLa cells were seeded in 96-well tissue
culture plates at a density of 8000 cells per well and maintained
overnight in a DMEM medium. Cells were then treated with various
concentrations of PFPDA (1, 5, 10, 20, 50, and 100 μM) respectively,
followed by incubation at 37 °C for 24 h. After discarding the culture
medium, the cells were then treated with 100 mL of MTT (1 mg/mL in
PBS) followed by incubation at 37 °C for 4 h. After the supernatant was
removed, the cells were lysed by adding 100 μL DMSO per well, and
the absorbance of the purple formazan was recorded at 520 nm using a
Spectra MAX 340PC plate reader.
Regulation of Fluorescence of PFPDA with MGCB: Malachite
green carbinolbase (MGCB) was dissolved in DMSO. A surfactant
cetyltrimethylammonium bromide (CTAB) (50 mM) was dissolved in
200 mM NaCl aqueous solution and the whole solution served as the
working system with a slightly acidic pH value. To a fluorimeter cuvette
with 1 mL CTAB (50 mM) in NaCl (200 mM) was added MGCB (100 μM)
and PFPDA (2 μM). The whole system was irradiated under 254 nm
UV light. The fluorescence of PFPDA was recorded under excitation at
390 nm.
Fluorescence Imaging of Autophagy in HeLa Cells: 20 μL of 1.0 mM
PFPDA and 13 μL rapamycin (3.8 mM in DMSO) was added into 2 mL
of serum-free DMEM medium containing HeLa cells in 35 mm × 35 mm
plate (the final concentration: [PFPDA] = 10 μM, [rapamycin] = 25 μM).
The plate was incubated at 37 °C for 8 h, then the medium was removed
and the cells were washed with phosphate buffered saline (PBS, pH 7.4)
twice. The fluorescent images were recorded on fluorescence microscopy
using a 380/30 nm excitation filter with 100 ms exposure time. Another
plate of HeLa cells as a control was treated in the same way except for
the addition of rapamycin.
Acknowledgements
The authors are grateful to the National Natural Science Foundation
of China (no. 21033010, 21003140, 90913014, and 21021091) and the
Major Research Plan of China (no. 2011CB932302, 2012CB932600, and
2011CB808400).
Received: July 28, 2012
Published online:
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DOI: 10.1002/adfm.201202132