A mitochondria-targeting fluorescent Fe3+ probe and its application in labile Fe3+ monitoring via imaging and flow cytometry

Article abstract of DOI:10.1016/j.dyepig.2018.05.008

The biogenesis of heme and iron-sulfur cluster in mitochondria is closely associated with the intracellular iron homeostasis. In this study, a mitochondria-targeting probe with the reversible turn-on fluorescent response to Fe³⁺ was developed for mitochondrial labile Fe³⁺ monitoring by modifying spirolactam rhodamine with a chelator. With this probe, the mitochondrial labile Fe³⁺ fluctuation in adherent cells upon ferric citrate incubation was visualzied via confocal imaging, and the flow cytometric assay for mitochondrial Fe³⁺ in suspension cells, which is difficult to be monitored via confocal imaging, was also developed. Moreover, the labile Fe³⁺ drop in mitochondria of K562 cells undergoing the DMSO-stimulated erythroid differentiation was observed for the first time.

Full text of DOI:10.1016/j.dyepig.2018.05.008

DyesandPigments157(2018)328–333
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A mitochondria-targeting fluorescent Fe3+ probe and its application in
labile Fe3+ monitoring via imaging and flow cytometry
Chengcheng Zhua, Mengjia Wanga, Lin Qiua,b,∗∗, Shuangying Haoc, Kuanyu Lic,∗∗∗, Zijian Guoa,
Weijiang Hea,∗
a
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou 213164, China
Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China
b
c
A B S T R A C T
The biogenesis of heme and iron-sulfur cluster in mitochondria is closely associated with the intracellular iron
homeostasis. In this study, a mitochondria-targeting probe with the reversible turn-on fluorescent response to
Fe3+ was developed for mitochondrial labile Fe3+ monitoring by modifying spirolactam rhodamine with a
chelator. With this probe, the mitochondrial labile Fe3+ fluctuation in adherent cells upon ferric citrate in-
cubation was visualzied via confocal imaging, and the flow cytometric assay for mitochondrial Fe3+ in sus-
pension cells, which is difficult to be monitored via confocal imaging, was also developed. Moreover, the labile
Fe3+ drop in mitochondria of K562 cells undergoing the DMSO-stimulated erythroid differentiation was ob-
served for the first time.
1. Introduction
intracellular iron homeostasis [4,8,9], although most iron exists in
bound state. However, the fluorescence sensing of Fe3+ is still chal-
lenging owning to the paramagnetic nature of Fe3+ quenching fluor-
escence critically. In fact, the fluorescent probe for mitochondrial labile
Fe3+ is rare [10], and the ROS probe dihydrorhodamine 123 has been
adopted for indirect sensing of mitochondrial labile iron fluctuation in
the study of Friedreich's ataxia, since Fe3+ induces ROS generation in
cells [11].
Iron plays essential roles in biological processes of eukaryotic cells
such as oxygen transport [1], electron transfer [2], and enzymatic
catalysis [3]. Most of these functions were associated with mitochon-
dria, which are proposed as iron reservoir and metabolism site in cells.
The biogenesis of heme and iron-sulfur clusters are the most important
iron-related events in mitochondria [4], and the Fe-S clusters are es-
sentially involved in Iron Regulatory Protein 1 (IRP1) which regulates
the intracellular iron homeostasis. In addition, the dysregulation of
mitochondrial iron has been reported to induce ROS generation and
mitochondrial membrane lipid peroxidation, which results in diseases
such as Parkinson's disease, Alzheimer's disease, and Friedreich's ataxia
[5]. The newly discovered type of cell death, ferroptosis, shows also the
increased mitochondrial membrane as one of its characteristics [6].
How to understand the physiological processes of iron homeostasis to
sustain mitochondrial functions demands reliable technique to sense
mitochondria iron perturbation. Since the isotope labelling and X-ray
related analysis are difficult to offer in situ iron information in live cells
[7], fluorescence imaging, which is able to offer in situ spatial-temporal
information of chemical species in live systems, has been proposed for
labile (or chelatable) Fe3+ monitoring in cells to evaluate the
In this study, a new rhodamine-based fluorescent probe for Fe3+
,
Mito-RhFe (Scheme 1), was developed for mitochondrial labile Fe3+
sensing in living cells. In this probe, spirolactam rhodamine was
adopted as fluorescence signaling group due to its turn-on response
triggered by target-induced ring opening process [12]. Moreover, the
delocalized positive charge of rhodamine is expected to favor the mi-
tochondrion targeting ability [13]. N2-hydroxyethyldiethylenetriamine
(HEDTA) was incorporated as a chelator due to its fine Fe3+ affinity
[14]. This new probe shows not only the Fe3+-specific turn-on fluor-
escent response but also the distinct mitochondrion target ability in live
cells. The mitochondrial labile Fe3+ imaging in adherent cells and flow
cytometric assay for labile Fe3+ in mitochondria of suspension cells
were realized with this probe.
∗
Corresponding author. State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
Corresponding author. School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou 213164, China.
Corresponding author. Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China.
∗∗
∗∗∗
E-mail addresses: linqiu@cczu.edu.cn (L. Qiu), likuanyu@nju.edu.cn (K. Li), heweij69@nju.edu.cn (W. He).
https://doi.org/10.1016/j.dyepig.2018.05.008
Received 10 February 2018; Received in revised form 5 May 2018; Accepted 5 May 2018
Availableonline07May2018
0143-7208/©2018ElsevierLtd.Allrightsreserved.

C. Zhu et al.
DyesandPigments157(2018)328–333
by chromatographic column (CH2Cl2:CH3OH = 20:1, v/v), and the
product Mito-RhFe was obtained as orange solid. Yield, 20%. 1H NMR
(500 MHz, CDCl3, δ, ppm): 7.87 (m, Ar-H, 2H), 7.43 (m, Ar-H, 4H), 7.08
(m, Ar-H, 2H), 6.38 (m, Ar-H, 8H), 6.28 (m, Ar-H, 4H), 3.34 (q,
J = 7.08 Hz, 16H), 3.11 (t, J = 4.56 Hz, 2H), 3.04 (t, J = 7.12 Hz, 4H),
2.19 (m, 6H), 1.17 (t, J = 7.00 Hz, 24H). 13C NMR (126 MHz, CDCl3, δ,
ppm): 167.56, 153.45, 153.24, 148.8, 132.06, 131.73, 128.88, 127.90,
123.74, 122.64, 108.20, 105.82, 97.88, 64.95, 58.81, 55.44, 51.77,
44.34, 38.16, 12.61. HR-MS (positive mode, m/z): Calcd. 996.5751,
found 996.5749 for [M+H]+
.
Scheme 1. Synthesis of probe Mito-RhFe. Reagents and conditions: (i) POCl3,
1, 2-dichloroethane, reflux, 4 h (ii) HEDTA, acetonitrile, RT, overnight.
2.3. Colocalization of Mito-RhFe with MitoTracker Deep Red 633 in MCF-
7 cells and HeLa cells
2. Experimental
MCF-7 and HeLa cells were cultured respectively in DMEM sup-
plemented with 10% FBS (fetal calf bovine serum) in an atmosphere of
5% CO2 and 95% air at 37°C. The cells were stained with Mito-RhFe
(10 μM, 0.5 h) in PBS (1 × ) at ambient temperature. The staining
medium was prepared by adding the stock solution of Mito-RhFe in
DMSO into PBS buffer to the desired concentration (10 μM) and the
maximum DMSO content in the final solution was lower than 0.2%.
After the first imaging, the cells were stained further with MitoTracker
Deep Red 633 (1 μM, 5 min) at ambient temperature for the second
imaging. All the imaging was carried out with dual channel model
2.1. Materials, general methods and instrumentations
Solvents and reagents were commercial available and used without
further purification. All solvents for spectroscopic study were of spec-
trum grade. Water for spectroscopic determination is the deionized
water from MilliQ system (> 18 MΩ). All fluorescence spectra were
recorded using Horiba Scientific Fluoromax-4 fluorescence spectro-
photometer, while the UV/Vis absorption spectra were recorded on a
LAMBDA-35 spectrophotometer. All NMR spectra were recorded with
Bruker DRX-500 or Bruker DRX-300. All the pH values were record
using a pH meter Sartorius PB-10 meter. High resolution mass spec-
trometric data were determined using an Agilent 6540 Q-TOF HPLC-MS
spectrometer. All the antibodies for western-blotting such as rabbit anti-
MFRN1 (Abcam), rabbit anti-MFRN2 (Abcam), rabbit anti-globin
(Abcam), mouse anti-GAPDH (proteintech) were commercial available.
HPLC analysis of Mito-RhFe (10 μM) in Tris-HCl buffer was carried out
with SHIMADZU LC-20AT using a AcclaimTM 120C18 column (5 μm,
120 Å, 4.6*250 mm). The column temperature was at 30°C, the UV
detection wavelength was 550 nm, and the mobile phase was the mixed
solvent of CH3OH and H2O (9:1, v/v).
(Mito-RhFe
channel:
bandpass
570–620 nm,
λex = 543 nm;
MitoTracker channel: bandpass 665–730 nm, λex = 633 nm). The ima-
ging was realized with a confocal laser scanning fluorescence micro-
scope (Zeiss LSM710).
2.4. Confocal fluorescence imaging for intracellular labile Fe3+ in living
cells
HeLa cells were cultured in DMEM supplemented with 10% FBS
(fetal calf bovine serum) in an atmosphere of 5% CO2 and 95% air at
37 °C. The cells were stained with Mito-RhFe (10 μM, 30 min at am-
bient temperature). After imaging, the cells were incubated further with
ferric citrate (20 μM) in PBS for 0.5 h. After the second imaging, the
cells were further incubated with TPEN (50 μM) in PBS for 0.5 h at room
temperature for imaging. The imaging was carried out using a confocal
2.2. Synthesis Mito-RhFe (Scheme 1)
2.2.1. Synthesis of rhodamine B chloride
fluorescence microscope (Zeiss LSM710) with
570–620 nm upon excitation at 543 nm.
a
bandpass of
Rhodamine B (1.0 g, 2.3 mmol) was dissolved in 1,2-dichloroethane
(12 mL) and POCl3 (0.6 mL) was added dropwise in 5 min. Then the
mixture was refluxed with stirring for 4 h. After being cooled to room
temperature, the mixture was evaporated in vacuo to remove solvent,
and the resulted residual can be utilized as the raw material for Mito-
RhFe preparation directly.
2.5. Flow cytometric assay for exogenous labile Fe3+ in MEL cells
MEL cells were seeded in 60 mm dishes with the number of 5 × 105
and cultured with DMEM supplemented with 10% FBS (fetal bovine
serum) in an atmosphere of 5% CO2 and 95% air at 37 °C for 24 h. Then
different concentration of ferric citrate (0, 10, 20, 40, and 50 μM) was
added respectively into the medium to culture the cells for 6 h. After
that, the cells were collected and rinsed with PBS for 3 times. Then the
cells were stained with Mito-RhFe (10 μM, 0.5 h) in PBS cooled by ice.
After removing the staining medium, the cells were rinsed by PBS for 3
times and resuspended in PBS for flow cytometric assay with a flow
cytometer (BD LSR Fortessa™) using PE channel.
2.2.2. Synthesis of 1-bis(1-aminoethyl)amino ethanol (HEDTA)
Diethylenetriamine (150 g, 1454 mmol) and concentrated sulfuric
acid (93 mL) were mixed in water followed by adding ethylene oxide
(30 g, 681.0 mmol) slowly in 1 h. Then the mixture was stirred at room
temperature overnight. The mixture was poured into 400 g NaOH so-
lution (50%), and the precipitate was filtered off. The filtrate was ex-
tracted with isopropyl alcohol, and the combined extracts were com-
bined and evaporated in vacuo. The residue was distilled in vacuo and
the fraction collected at 148°C/11 mmHg is the final product. Yield,
13%. 1H NMR (400 MHz, CDCl3, δ, ppm): 3.35 (t, J = 4.0 Hz, 2H), 2.52
(t, J = 6.0 Hz, 4H), 2.37 (t, J = 4.0 Hz, 2H), 2.33 (t, J = 6.0 Hz, 4H),
1.99 (br, 4H).
2.6. Flow cytometric assay for endogenous labile Fe3+ in stimulated
K562 cells
The cells were seeded in 35 mm dishes with a density of 1 × 104 per
dish and cultured for 4 days in RPMI 1640 medium (Hyclone, USA)
containing 2% DMSO (Sigma, v/v), 10% fetal bovine serum (FBS), 100
U/ml penicillin and 100 μg/mL streptomycin at 37 °C in incubator
containing a humid atmosphere of 5% CO2 and 95% air. The medium
was replaced every two days, and 50 μM ferric citrate was added to the
medium on second day after each replacement. Then the cells were
treated respectively for Western blot analysis and flow cytometric
2.2.3. Synthesis of Mito-RhFe
Rhodamine B chloride (1.07 g, 2.3 mmol) was dissolved in acet-
onitrile, and HEDTA (5 mL in 20 mL acetonitrile) was added dropwise
into the solution in 1 h at 0 °C. The resulted mixture was stirred at room
temperature overnight. Then the solvent was removed in vacuo.
Washing the residue with water (20 mL × 4), and the solid was purified
329

C. Zhu et al.
DyesandPigments157(2018)328–333
500
400
300
200
100
0
Fig. 1. Emission (a, λex = 540 nm) and absorp-
tion (b) spectra of Mito-RhFe (10 μM) in Tris-
HCl buffer (20 mM, pH 7.20, 50% methanol, v/
v) upon titration with Fe3+ (0–20 equiv). Inset in
(a): Titration profile according to the emission at
580 nm. Inset in (b): photograph of Mito-RhFe
solution before (left) and after (right) Fe3+ ad-
dition.
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(b)
(a)
Fe3+
700
600
500
400
300
200
100
0
0
5
10 15 20 25 30 35 40
[F3e+3+]/L2
[Fe ]/[Mito-RhFe
]
550
600
650
700
200
300
400
500
600
Wavelength (nm)
Wavelength (nm)
assay. For Western blot analysis, the cells were suspended in RIPA lysis
buffer to quantify the target protein expression use 12% SDS-PAGE gels.
The antibodies were used following the user guide. The flow cytometric
assay was carried out in the procedure described in 2.5. The control
without 2% DMSO was also carried out for comparison.
with the turn-on response of Mito-RhFe to Fe3+, except for Cd2+
Zn2+, and Ni2+ reducing the Fe3+-induced emission enhancement
factors respectively to ∼58-, 50-, and 40-fold. In addition, the Fe3+
,
-
induced emission enhancement of Mito-RhFe can be reduced distinctly
by adding TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine,
20 equiv), a cell membrane permeable transition metal chelator
(Fig. 2b). The second Fe3+ addition to probe solution shows again the
enhanced emission similar to that upon first addition. Therefore, the
Fe3+ sensing behavior of probe Mito-RhFe is reversible in general. This
reversible response has also been confirmed by HPLC analysis. It was
found that Mito-RhFe showed only one signal of retention time
6.88 min for the spirolactam form (100% area), and Fe3+ addition led
to the second signal of retention time 5.44 min besides the signal of the
spirolactam form. Considering the turn-on response in UV-vis and
emission spectra induced by Fe3+, this new signal could be assigned to
the ring open form of Mito-RhFe bound with Fe3+. The subsequent
treatment with TPEN to remove Fe3+ made almost all the new signal
transit to the former signal for spirolactam form, demonstrating the
recovery of spirolactam from the ring open form (Fig. S5). The fluor-
escence detection limit of this probe is determined as 1.07 × 10−7 M
(Fig. S6) via titration experiment. In addition, the turn-on response of
this probe can be fulfilled with Fe3+ in ∼200 s (Fig. S7). In the pH
range from 6.0 to 12.3, this probe shows almost no fluorescence (Fig.
S8), and the distinct fluorescence enhancement of Mito-RhFe only can
be observed when medium pH is lower than pH 5.0. This implies that
the ring opening process of this spirolactam probe occurs only at
pH < 5.0. The pH-independent stability of this spirolactam probe in
pH 6.0–12.3 suggests Mito-RhFe is suitable for Fe3+ sensing in mi-
tochondria, which possesses a slightly basic microenvironment of pH of
∼8.0, providing the colocalization experiment disclosing the mi-
tochondrion-preferring distribution of Mito-RhFe (vide infra).
3. Results and discussion
3.1. Fluorescent response of probe Mito-RhFe to Fe3+ in solution
The solution of compound Mito-RhFe (10 μM) in Tris-HCl buffer
(20 mM, pH 7.2, 50% methanol, v/v) displays almost no fluorescence
but the typical absorption bands at 205, 250 and 355 nm of spirlo-
lactam form for rhodamine. The rhodamine emission band centered at
578 nm appears and increases with a ∼8 nm bathochromic shift upon
Fe3+ titration and excitation at 540 nm (Fig. 1a). The fluorescence
enhancement factor, (F-F0)/F0, attains to ∼90-fold upon titration with
20 equiv of Fe3+. In UV-vis Fe3+ titration of this probe, the absorption
spectra show the distinct drop of its absorption band at 355 nm, and the
increase of new bands centered respectively at 273 and 314 nm.
Moreover, an absorption band at 555 nm appears distinctly with a
shoulder at 517 nm (Fig. 1b). This band is the characterized absorption
of rhodamine in ring open form. In the meantime, the color of Mito-
RhFe solution changes from colorless to magenta (Fig. 1b inset). All
these phenomena are consistent with the expected conversion of Mito-
RhFe from spirolactam to ring opening form.
The fluorescent response of Mito-RhFe (10 μM) to metal cations of
interest has been investigated via determining the emission spectra of
Mito-RhFe (Fig. 2a). The spectra indicate that most of the tested metal
cations such as Fe2+, Hg2+, Cd2+, Zn2+, Pb2+, Co2+, Ag+, Cu2+
,
Mn2+, Ni2+ (20 equiv) and K+, Mg2+,Ca2+ and Na+ (100 equiv) do
not induce the fluorescence of Mito-RhFe, yet Fe3+, Cr3+ and Al3+ (20
equiv) increase the probe fluorescence with the enhancement factors of
90-, 6-, and 14-fold, respectively. With the scarcity of Cr3+ and Al3+ in
living systems, the emission enhancement induced by Cr3+ and Al3+
does not affect the Fe3+ sensing behavior of Mito-RhFe in cells.
Moreover, the presence of most tested metal cation does not interfere
3.2. Fluorescence imaging of mitochondrial labile Fe3+ in live cells via
Mito-RhFe staining
Fluorescence imaging of Mito-RhFe has been investigated in live
cells such as MCF-7 and HeLa cells with a confocal laser scanning
140
Fig. 2. (a) Fluorescent response of Mito-RhFe
(10 μM) to Fe2+ Hg2+ Cd2+ Zn2+ Pb2+
, , , , ,
Mito-RhFe+Metal
Mito-RhFe+Fe3+
(a)
(b)
600
500
400
300
200
100
0
Mito-RhFe +Metal+Fe3+
Co2+, Ag+, Cu2+, Mn2+, Ni2+ (200 μM), and
K+, Mg2+,Ca2+, and Na+ (1000 μM) in Tris-HCl
buffer (20 mM, pH 7.20, 50% methanol, v/v)
with (grey) or without (black) the presence of
Fe3+ (200 μM). (F-F0)/F0 was calculated based
on the emission at 580 nm. (b) Fluorescence
spectra of Mito-RhFe (10 μM) obtained as free
probe, in the presence of 20 equiv Fe3+, and
after the subsequent TPEN (200 μM) addition.
120
100
80
60
40
20
0
Mito-RhFe+Fe3++TPEN
Mito-RhFe
λ
ex = 540 nm.
550
600
650
700
+
+
+
2+
2+
2
2+
86.8364.5377601.269
2
-5
+
03.926-504.326
2
8
+
-06.42600.576-605.355-609.218
2
06
+
.9431234.082905.130509.8731342.29389
3+
2
3+
3+
2+
Wavelength (nm)
Al
Fe
Zn Mix
Pb
Ag
Cr
Hg
Co
Mn Ni
Cu
Fe
Cd
330

C. Zhu et al.
DyesandPigments157(2018)328–333
Fig. 3. Colocalization of Mito-RhFe with MitoTracker Deep
red 633 in MCF-7 cells. The cells were stained firstly by
10 μM probe in PBS for 30 min at 37 °C. After the first ima-
ging, the cells were incubated further with 1 μM MitoTracker
Deep Red 633 (5 min) in PBS at 37 °C for the second imaging.
(a) fluorescence image of cells recorded in first imaging with
a bandpass of 570–620 nm upon excitation at 543 nm; (b)
fluorescence image of cells in (a) recorded in second imaging
with a bandpass of 665–730 nm upon excitation at 633 nm
(c) Overlay of (a) and (b). (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to
the Web version of this article.)
microscope (Zeiss LSM710). The imaging shows the weak fluorescence
in cytosol after 30 min of incubation with Mito-RhFe, while cells
without probe incubation show no fluorescence in the same condition.
This indicates the fine cell membrane permeability of this probe.
Colocalization with MitoTracker Deep Red 633 in MCF-7 cells discloses
that the intracellular fluorescence of Mito-RhFe recorded in probe
channel is localized on mitochondria visualized by MitoTracker fluor-
escence recorded in MitoTracker channel (Fig. 3). The Pearson's colo-
calization coefficient of Mito-RhFe with MitoTracker was 0.96. Colo-
calization in HeLa cells discloses also the mitochondria-targeting ability
with a coefficient of ∼0.90.
With the mitochondrion targeting ability, Mito-RhFe was utilized to
visualize mitochondrial labile Fe3+ fluctuation in HeLa cells upon Fe3+
incubation by imaging (Fig. 4). The confocal imaging displays the weak
fluorescent dots (Fig. 4b), indicating the low mitochondrial labile Fe3+
level. After being incubated with ferric citrate (20 μM) for 30 min, the
cells show the distinctly enhanced fluorescence (Fig. 4c), suggesting the
labile Fe3+ level in mitochondria can be effectively enhanced by exo-
genous Fe3+ incubation. The subsequent incubation with TPEN makes
the fluorescence in mitochondria even lower than that shown in Fig. 4b.
This demonstrates the labile Fe3+ in mitochondria can be effectively
removed by TPEN scavenging (Fig. 4d). All these indicate that the labile
Fe3+ fluctuation in mitochondria can be effectively monitored in a
reversible manner by Mito-RhFe.
concentration if the ferric citrate concentration attains to 20 μM
(Fig. 5a), although 10 μM ferric citrate incubation induces no difference
from the control without Fe3+ incubation. The median fluorescence
determined at different ferric citrate concentration shows also that
ferric citrate incubation will increase the labile Fe3+ level in mi-
tochondria of MEL cells (Fig. 5b). All the results imply Mito-RhFe can
be practically applied in sensing mitochondrial labile Fe3+ in suspen-
sion cells via flow cytometry.
Erythroid differentiation is essential for erythropoiesis from hema-
topoietic stem cell [15], and artificial stimulated erythroid differ-
entiation is normally adopted in lab to explore the complicated phy-
siological network involved erythropoiesis. The differentiation is
usually accompanied by distinct hemoglobin synthesis and upregula-
tion of the mitochondrial iron importing protein Mitoferrin-1 (MFRN1)
containing Fe-S cluster [4,16], and maturation is normally confirmed
by benzidine staining to show the distinctly upregulated level of he-
moglobin [7a,17]. Since hemoglobin and Fe-S cluster biogenesis in er-
ythropoiesis is closely associated with mitochondrial iron accumulation
and incorporation [4a,18], the labile Fe3+ perturbation in mitochon-
dria can be expected. The success of flow cytometric assay of mi-
tochondrial labile Fe3+ via Mito-RhFe staining inspires us to explore
the possibility to monitor erythroid differentiation process via sensing
mitochondria labile Fe3+ levels in the stimulated leukemic cells. The
human K562 erythroleukemia cells, the common model for erythroid
progenitors upon stimulation with many stimulatory factors such as
DMSO [19,20], were cultured in medium with 2% DMSO for 4 days for
labile Fe3+ flow cytometric assay. The results disclose that the median
fluorescence of the DMSO-stimulated cells is distinctly decreased when
compared with that of the control without DMSO stimulation (Fig. 6a).
On the other hand, the western blot results show that DMSO stimulation
leads to the distinct hemoglobin expression in the cells, while the
control without DMSO induction shows almost no this protein (Fig. 6b).
In addition, the obviously enhanced MFRN1 was also observed upon
DMSO stimulation. All the western blot results confirm the erythrocyte
maturation stimulated by culturing K562 cells for 4 days in DMSO-
containing RPMI-1640 medium. The accompanied median fluorescence
decrease in flow cytometric assay suggests the stimulated synthesis of
heme and Fe-S cluster in mitochondria may consume too much iron in
mitochondria which decreases the labile Fe3+ level in mitochondria,
and the mitochondrial labile Fe3+ monitoring via flow cytometry might
be an effective tool for the assessment of erythroid differentiation.
3.3. Flow cytometric assay for mitochondrial labile Fe3+ in suspension cells
via Mito-RhFe staining
With the successful imaging for labile Fe3+ in adherent cells, Mito-
RhFe was further investigated for its flow cytometric application in
sensing mitochondrial labile Fe3+ in suspension cells. Since the sus-
pension cells, such as the blood cells, can't be monitored by confocal
fluorescence imaging, the flow cytometric assay for labile Fe3+ instead
of confocal imaging becomes the promising alternative for fluorescence
sensing of labile Fe3+ in suspension cells, especially the mitochondrial
iron metabolism is of great significance for blood cells. Therefore mouse
erythroleukaemia (MEL) cells were incubated respectively with ferric
citrate solutions of different concentration (0–50 μM, 6 h at 37 °C) fol-
lowed by staining with probe solution (10 μM, 30 min at 0 °C). The
stained cells were determined by a flow cytometer using a PE channel.
The determined cell population distribution discloses the fluorescence
for population maximum increases distinctly with the ferric citrate
Fig. 4. Confocal fluorescence imaging of HeLa
cells stained by Mito-RhFe (10 μM in PBS,
30 min at 37 °C). (a) Bright field cell image; (b)
fluorescence image of the stained cells; (c)
fluorescence image of the cells in (b) incubated
further with ferric citrate (20 μM, 30 min at
37 °C); (d) fluorescence image of cells in (c)
treated further with TPEN (50 μM, 30 min at
37 °C) incubation.
560–620 nm. Scale bar, 10 μm.
λex = 543 nm, bandpass:
331

C. Zhu et al.
DyesandPigments157(2018)328–333
Fig. 5. Flow cytometric assay of MEL cells in-
cubated with ferric citrate solutions of different
concentration for 6 h (37 °C), followed by in-
cubation with Mito-RhFe (10 μM, 30 min, 0 °C).
Test was realized with
a PE channel. (a)
Histogram of MEL cells incubated with Fe3+ of 0
(red), 10 (blue), 20 (green), 40 (yellow), and 50
(purple) μM. (b) Median fluorescence of the
stained MEL cells induced by Fe3+ incubation
shown in (a). (For interpretation of the refer-
ences to color in this figure legend, the reader is
referred to the Web version of this article.)
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Acknowledgements
We thank the financial supports from the National Basic Research
Program of China (No. 2015CB856300), National Natural Science
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.dyepig.2018.05.008.
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