Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures
Aim. To study possibile application of C2, C9, C18 and JC-1 carbocyanine fluorescent dyes for cell culture characterization. Methods. Morphological methods, fluorescence-activated cell sorting (FACS) analysis, luminescent microscopy were used. Results. The studied carbocyanine probes were shown to b...
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Goncharuk, E.I. Borovoy, I.A. Pavlovich, E.V. Malyukin, Yu.V. Grischenko, V.I. 2019-06-14T18:14:46Z 2019-06-14T18:14:46Z 2009 Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures / E.I. Goncharuk, I.A. Borovoy, E.V. Pavlovich, Yu.V. Malyukin, V.I. Grischenko // Biopolymers and Cell. — 2009. — Т. 25, № 6. — С. 484-490. — Бібліогр.: 17 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0007FB https://nasplib.isofts.kiev.ua/handle/123456789/153846 57.085.2:577.336 Aim. To study possibile application of C2, C9, C18 and JC-1 carbocyanine fluorescent dyes for cell culture characterization. Methods. Morphological methods, fluorescence-activated cell sorting (FACS) analysis, luminescent microscopy were used. Results. The studied carbocyanine probes were shown to be preserved in dividing cells for at least 4 duplications. It was found that carbocyanine probe JC-1 did not transit from cell to cell under combined culturing of labeled and non-labeled cells. Conclusions. The paper covers the use of carbocyanine fluorescent probes for long-term culturing of cell lines. Probes C9 and JC-1 were optimal for the proliferative culture observation, allowing to trace mitochondrial functional state. Цель. Исследовать возможности применения карбоцианиновых флуоресцентных зондов С2, С9, С18 и JC-1 для характеристики культур клеток. Методы. Использованы морфологи- ческие методы, метод проточной цитофлуориметрии (FACS- анализ), люминесцентная микроскопия. Результаты. Показано, что исследуемые карбоцианиновые зонды сохраняются в делящихся клетках в течение не менее четырех удвоений. Установлено, что карбоцианиновый зонд JC-1 не переходит из клетки в клетку при совместном культивировании меченых и немеченых клеток разных культур. Выводы. Определено, что указанные флуоресцентные зонды можно использовать при долговременном культивировании клеточных линий. Для наблюдения за пролиферирующими культурами оптимальным является применение зондов С9 и JC-1, что позволяет отслеживать функциональное состояние митохондрий. Mета. Дослідити можливості застосування карбоціанінових флуоресцентних зондів С2, С9, С18 та JC-1 для характеристики культур клітин. Методи. Використано морфологічні методи, метод проточної цитофлуориметрії (FACS-аналіз), люмінесцентну мікроскопію. Результати. Показано, що досліджені карбоціанінові зонди зберігаються в клітинах, що діляться, протягом не менш чотирьох подвоєнь. Встановлено, що карбоціаніновий зонд JC-1 не переходить із клітини в клітину при одночасному культивуванні мічених і немічених клітин різних культур. Висновки. Встановлено, що зазначені флуоресцентні зонди можна використовувати при довготривалому культивуванні клітинних ліній. Для спостереження за проліферуючими культурами оптимальним є застосування зондів С9 і JC-1, що дозволяє відслідковувати функціональний стан мітохондрій. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Біоорганічна хімія Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures Новосинтезовані карбоцiанінові флуоресцентні зонди, їхня характеристика та поведінка в проліферуючих культурах Новосинтезированные карбоцианиновые флуоресцентные зонды, их характеристика и поведение в пролиферирующих культурах Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures |
| spellingShingle |
Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures Goncharuk, E.I. Borovoy, I.A. Pavlovich, E.V. Malyukin, Yu.V. Grischenko, V.I. Біоорганічна хімія |
| title_short |
Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures |
| title_full |
Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures |
| title_fullStr |
Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures |
| title_full_unstemmed |
Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures |
| title_sort |
newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures |
| author |
Goncharuk, E.I. Borovoy, I.A. Pavlovich, E.V. Malyukin, Yu.V. Grischenko, V.I. |
| author_facet |
Goncharuk, E.I. Borovoy, I.A. Pavlovich, E.V. Malyukin, Yu.V. Grischenko, V.I. |
| topic |
Біоорганічна хімія |
| topic_facet |
Біоорганічна хімія |
| publishDate |
2009 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Новосинтезовані карбоцiанінові флуоресцентні зонди, їхня характеристика та поведінка в проліферуючих культурах Новосинтезированные карбоцианиновые флуоресцентные зонды, их характеристика и поведение в пролиферирующих культурах |
| description |
Aim. To study possibile application of C2, C9, C18 and JC-1 carbocyanine fluorescent dyes for cell culture characterization. Methods. Morphological methods, fluorescence-activated cell sorting (FACS) analysis, luminescent microscopy were used. Results. The studied carbocyanine probes were shown to be preserved in dividing cells for at least 4 duplications. It was found that carbocyanine probe JC-1 did not transit from cell to cell under combined culturing of labeled and non-labeled cells. Conclusions. The paper covers the use of carbocyanine fluorescent probes for long-term culturing of cell lines. Probes C9 and JC-1 were optimal for the proliferative culture observation, allowing to trace mitochondrial functional state.
Цель. Исследовать возможности применения карбоцианиновых флуоресцентных зондов С2, С9, С18 и JC-1 для характеристики культур клеток. Методы. Использованы морфологи- ческие методы, метод проточной цитофлуориметрии (FACS- анализ), люминесцентная микроскопия. Результаты. Показано, что исследуемые карбоцианиновые зонды сохраняются в делящихся клетках в течение не менее четырех удвоений. Установлено, что карбоцианиновый зонд JC-1 не переходит из клетки в клетку при совместном культивировании меченых и немеченых клеток разных культур. Выводы. Определено, что указанные флуоресцентные зонды можно использовать при долговременном культивировании клеточных линий. Для наблюдения за пролиферирующими культурами оптимальным является применение зондов С9 и JC-1, что позволяет отслеживать функциональное состояние митохондрий.
Mета. Дослідити можливості застосування карбоціанінових флуоресцентних зондів С2, С9, С18 та JC-1 для характеристики культур клітин. Методи. Використано морфологічні методи, метод проточної цитофлуориметрії (FACS-аналіз), люмінесцентну мікроскопію. Результати. Показано, що досліджені карбоціанінові зонди зберігаються в клітинах, що діляться, протягом не менш чотирьох подвоєнь. Встановлено, що карбоціаніновий зонд JC-1 не переходить із клітини в клітину при одночасному культивуванні мічених і немічених клітин різних культур. Висновки. Встановлено, що зазначені флуоресцентні зонди можна використовувати при довготривалому культивуванні клітинних ліній. Для спостереження за проліферуючими культурами оптимальним є застосування зондів С9 і JC-1, що дозволяє відслідковувати функціональний стан мітохондрій.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/153846 |
| citation_txt |
Newly synthesized carbocyanine fluorescent probes, their characteristics and behavior in proliferating cultures / E.I. Goncharuk, I.A. Borovoy, E.V. Pavlovich, Yu.V. Malyukin, V.I. Grischenko // Biopolymers and Cell. — 2009. — Т. 25, № 6. — С. 484-490. — Бібліогр.: 17 назв. — англ. |
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| fulltext |
БІООРГАНІЧНА ХІМІЯ
Newly synthesized carbocyanine fluorescent
probes, their characteristics and behavior in
proliferating cultures
E. I. Goncharuk, I. A. Borovoy1, E. V. Pavlovich, Yu. V. Malyukin1, V. I. Grischenko
Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine
23, Pereyaslavskaya Str., Kharkiv, Ukraine, 61015
1State Scientific Institution «Institute for Single Crystals» NAS of Ukraine
60, Lenin Ave.,Kharkiv, Ukraine, 61001
lenapavlovich@gmail.com
Aim. To study possibile application of C2, C9, C18 and JC-1 carbocyanine fluorescent dyes for cell culture
characterization. Methods. Morphological methods, fluorescence-activated cell sorting (FACS) analysis,
luminescent microscopy were used. Results. The studied carbocyanine probes were shown to be preserved
in dividing cells for at least 4 duplications. It was found that carbocyanine probe JC-1 did not transit from
cell to cell under combined culturing of labeled and non-labeled cells. Conclusions. The paper covers the
use of carbocyanine fluorescent probes for long-term culturing of cell lines. Probes C9 and JC-1 were
optimal for the proliferative culture observation, allowing to trace mitochondrial functional state.
Key words: fluorescence, probes, human fibroblasts, cell culture.
Introduction. Investigations of the cell cultures func-
tioning require informative express methods. One of
them is the labeling of different cell components with
luminescent probes. It is evident that an agent
introduced into a cell should be non-toxic and affect
minimally the vital cell processes. Long-term stay of a
fluorescent label inside a cell is also desirable. The
dyes with the abovementioned properties may be used
for diagnostics of the cell culture state under various
physical and chemical influences. A next prospect is
the coding of biological samples, e. g. during their sto-
rage in cryobanks or when transporting them.
In addition, the cytological and histological studies
for tracing the fate of transplanted cell in a recipient’s
organism require the use of long-living probes, provi-
ding the information on the processes occurring in cells
and tissues during their vital activity.
We have synthesized and investigated the carbo-
cyanine dyes differing in their hydrophilic-hyd-
rophobic features. As it has been reported [1] the
staining with these dyes was performed prior to investi-
gating and there are only single papers on culturing cell
lines with integrated dyes [2, 3]. The research was ai-
med at comparative characterization of the behavior of
syn- thesized by us probes in proliferating cell cultures.
The study comprised the examining of localization
of probes in a cell, duration of their storage and inves-
tigation of the possibility of free intercellular transition
of the probes during combined culturing of two cell
lines.
Materials and Methods. Fluorescent probes C2
(3,3'-diethyloxacarbocyanine bromide), C9 (3,3'-dide-
484
ISSN 0233-7657. Biopolymers and Cell. 2009. Vol. 25. N 6
Institute of Molecular Biology and Genetics NAS of Ukraine, 2009
cyloxacarbocyanine bromide), С18 (3,3'-dioctadecyl-
oxacarbocyanine bromide) and JC-1 (5,5',6,6'-tetra-
chlor-1,1',3,3'-tetraethyl-benzoimidazolylcarbocyani-
ne iodide) were synthesized at the Institute of Scin-
tillation Materials of the State Scientific Institution
«Institute for Single Crystals» NAS of Ukraine. When
staining the cell we used solutions of probes with con-
centration from 0.1 µM to 100 µM.
The behavior of probes has been studied in the cells
of human fibroblast (HF) diploid line and in the cells of
porcine embryo kidney (SPEV). The cells were cultu-
red in Dulbecco’s modified Eagle’s Medium (DMEM)
(Sigma) with adding 10 % fetal calf serum (FCS)
(HyClone) [4]. The number of viable cells was counted
on staining with trypan blue supravital dye. The probes
were integrated into the cell monolayer and cell sus-
pension, obtained enzymatically with following wash-
ing-out with Hank’s solution. The cells in suspended
state were incubated with probes for 15 min, afterwards
a non-bound dye was washed-out by adding Hank’s
solution into suspension in 9:1 ratio and centrifuged at
1,000 rot/min.
Afterwards the stained with fluorescent dyes cells
were cultured at 37 °C and 5 % CO2 [5]. The distri-
bution of dye in cells was assessed using inverted
fluorescent microscope Olympus IX71 with digital
camera Olympus C-5060, the excitation band was 450–
490 nm. Probe toxicity was tested by morphology and
proliferative activity of probe-labeled cells within in
vitro experiments.
The flow cytometry investigations were performed
with FACS Calibur cytofluorimeter (Becton-Dickin-
son, USA) using reagents of the same company. Prior
to the analysis the cells were suspended, washed-out
from nutritive medium and placed into isotonic solu-
tion. The cells were incubated with working concen-
trations of the probes within 15 min. The results ob-
tained by flow cytometry were analysed with Win MDI
v.2.8 software and presented as point graphs.
To find out whether the adherence of fluorescent
probes is potential-dependent, the proton translocator
carbonylcyanide p-trifluoromethoxyphenylhydrazone
(FCCP) was used [6]. FCCP at of 5 µM concentration
was added to a proliferating cell culture after staining
with fluorescent probes. The luminescence changes in
cells were estimated by luminescent microscopy.
To induce apoptosis, 10 µM etoposide was intro-
duced into the medium for 24 h with subsequent dou-
ble washing-out.
To examine the possibility of transition of fluo-
rescent probes from stained cells into non-stained ones
a method of co-culturing was applied. In the first case
the cells of HF culture were stained and the SPEV were
not, in the other case – vice-versa. After staining, the
cells were plated together with non-stained ones into
one flask on a glass and cultured for 2–3 days at 37 oC
and 5 % CO2. Distribution of dye in the cells was
evaluated by luminescent microscopy.
For the assessment of survival duration of the fluo-
rescent probes in the proliferating culture stained with
fluorescent dyes, the cells were cultured under standard
conditions for 10 days. During this term the presence
and intensity of luminescence were examined.
Results. Carbocyanine dyes have been widely used
in fluorescent labeling of cells and tissues since the late
70 s. Iodides and perchlorates have been used as coun-
ter-ions. The synthesized by us dyes C2, C9 and C18
differed in using Br-anions for this purpose. Herewith
the C2 dye is well soluble in water, and the probe C18
is poorly soluble, but due to its highly hydrophobic
behavior it integrates into membrane lipid areas. This
difference in hydrophilic-hydrophobic properties is
determined by the length of alkyl substituents at nitro-
gen atom of oxazole cycle. The dye C9 was synthesized
additionally as a probe with intermediate properties
due to the presence of alkyl «tails», consisting of 9 car-
bon atoms in the molecule. The JC-1 dye was obtained
by the method [7]. This dye exists in two forms: mono-
meric, fluorescing at 527 nm (green fluorescence) and
as J-aggregates with emission at 590 nm (orange fluo-
rescence) with 490 nm excitation wave length (Fig. 1).
The dyes C2, C9, C18 were synthesized according
to the methods [8]. The probes structures are shown on
the scheme.
Absorption and luminescence spectra of probes C2,
C9 and C18 measured in chloroform are identical
(within the measurement conditions) for all the dyes,
with fluorescence maximum at 515 nm (Fig. 2).
At the first stage of research the influence of carbo-
cyanine probes on the morphology and proliferative
activity was examined in vitro. During incubation of
the cells within the media with various content of fluo-
485
NEWLY SYNTHESIZED CARBOCYANINE FLUORESCENT PROBES
rescence probes, the optimal dye concentrations were
found for the studied cultures. For the probe C2 it was
1 µM, 10 µM – for the C9 and JC-1, 100 µM – for the
C18 dye. At optimal probe concentrations (and lower)
the morphology of human fibroblasts and SPEV cells
remained unchanged as compared to these indices for
not-labeled cells.
When increasing the concentration of probes in cell
suspensions, a reduction of cell viability was found. At
lower concentration of the dyes just a slight lumines-
cence of cell organelles was observed.
Fig. 3, A (see inset) shows the HF cells labeled with
fluorescent probe C2. As the figure demonstrates, the
dye is localized in filamentary structures with the
length up to 60 nm, the structures are located in
cytoplasm evenly, non-stained nucleus is soundly
manifested. There was observed quite evident flash
inside the whole cell volume that testifies to the fact of
unstable binding of the probe with organelles. During
the staining of cell suspension with the probe C9 (Fig.
3, B, see inset) its distribution in the cell cultures was
analogous, however the con- tours of mitochondria
were distinct, the baseline light-striking was absent in
the visible field.
The cells of HF cultures stained with the C18 dye
are presented in Fig. 3, C (see inset). The distribution of
this dye in fibroblast cells differs from that for the C2
and C9 probes. The luminescence was of dotty cha-
racter, the staining of mitochondria filamentary struc-
tures was absent.
486
GONCHARUK E. I. ET AL.
100
80
60
40
20
0
F
lu
o
re
sc
en
ce
,a
.u
.
400 500 600 700
Wave length, nm
JC-1
Water-DMSO
Fig. 1. Fluorescence spectrum of JC-1 dye in dimethyl sulfoxide
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
In
te
n
si
ty
,a
.u
.
425 450 475 500 525 550 575 600 625
Wave length, nm
Fluorescence
Excit, 470 nm
CHCl3
Absorption in
CHCl3
Fig. 2. Absorption and luminescence spectra of C2, C9, C18 dyes
N
O O
N
Probe C2
C2H5
C2H5
Br–
+
O
NN
O
C9H19
C9H19
Br–+
O
NN
O
C18H37
C18H37
Br–+
Probe C18
N
NN
N
C2H5
+
C2H5
C2H5 C2H5
Cl
Cl
Cl
Cl
Br–
Probe JC-1
Probe C9
Scheme. Structure of the probes
When staining the cells with the probe JC-1, there
was revealed a picture morphologically similar to those
for the dyes C2 and C9, exept for the fact that lumines-
cence of the cell organelles was observed in the orange
area (Fig. 4, see inset). There was observed green lumi-
nescence, associated with organelles, in cytoplasm.
This picture testifies to the presence of the dye in the
cells in form of J-aggregates.
The character of distribution of the dyes C2 and C9
in the SPEV culture cells differs from that in the HF
cells. Separated mitochondria, evenly distributed along
the whole SPEV cell, were stained (Fig. 5, A, see in-
set). Visually the SPEV cell chondriome did not differ
significantly from filamentary one of human fibro-
blasts (Fig. 5, A, B, see inset). As for the probe C18, its
localization in SPEV cells was similar to that in fib-
roblasts.
Information about the interaction of probes with
cells was also obtained by FACS method. The results
of flow cytometry of HF culture stained with the carbo-
cyanine probes are shown in Fig. 6. Each graph point
corresponds to a cell. The cell coordinates are the inten-
sities of fluorescence at the 520 ± 15 nm (FL1-H) and
580 ± 20 nm (FL2-H) fluorescence channels. R1 and
R2 are cell regions, limited by the parameter of fluo-
rescent intensity at the channels.
Cell fluorescence of HF culture stained with the
probe C2 as well as that of the culture stained with the
probe C9 is in a green fluorescence area, region (λ =
= 530 nm) (Fig. 6, A, B).
Fluorescence of the probe C18 in contrast to the
C2 and C9 probes is in the area of red fluorescence (λ =
= 670 ± 20 nm) and the cells are distributed into two
groups (Fig. 6, C).
Flow cytometry analysis of the HF cell culture
stained with the probe JC-1 has shown that the fluores-
cence in the orange region (λ = 585 ± 20 nm) is inhe-
rent for the case under study, moreover the cells are
distributed into two groups (Fig. 6, D). In this area J-
aggregates, formed on mitochondria with a high trans-
membrane potential, are luminescent. We induced apo-
ptosis in these cells using etoposide. In this case the
shift of fluorescence into green area was observed.
Thus, in fibroblasts the dyes were localized in fi-
lamentary structures located along the whole cell, si-
milar to the system of fibroblast mitochondria–chond-
riome described in the paper [9]. The JC-1 probe is a
well-known mitochondrial dye [10]. Its specific bin-
ding was proved using FCCP proton translocator. At
the potential alteration, orange luminescence changed
to green one as a result of the dye transition from poly-
meric to monomeric form.
When analogous manipulation was performed with
the cells of HF cultures, stained with other presumably
potential-dependent dyes, their release into the cell
cytoplasm and into environment was established in all
cases, as well as luminescent quenching as a result of
changing a chondriome membrane.
A potential of application of the carbocyanine pro-
bes for tracing the fate of some cells during long-term
culturing was studied. To investigate a possibility of
spontaneous dye transfer from cell to cell in prolifera-
ting culture the SPEV and HF cultures were co-cultu-
red. A significant difference in the morphology of fib-
roblast chondriome and kidney cells allowed us to per-
form the experiment, which we called «in vitro trans-
plantation».
It has been shown that during co-culturing the
C2-stained SPEV and non-stained HF cells, the dye
transfer from stained into non-stained cells takes place.
487
NEWLY SYNTHESIZED CARBOCYANINE FLUORESCENT PROBES
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Fig. 6. Cytofluorimetric analysis of cells stained with fluorescent
probes: A – C2: FL1-H (X-axis) and FL2-H (Y-axis), R1 = 31.95 %,
R2 = 51.89 %; B – C9: FL1-H and FL2-H, R1 = 98.1 %; C – C18:
FL1-H and FL2-H, R1 = 52.51 %, R2 = 28.91 %; D – JC-1: FL1-H
and FL2-H, R1 = 19.03 %, R2 = 79.72 %
Fig. 7 (see inset) shows bright luminescence of both
SPEV cells and fibroblasts for typical chondriome..
The same situation was observed during staining
the HF cells with the probe C2. The brightly lumines-
cent stained fibroblasts were seen against the back-
ground of non-stained SPEV cells. The luminescent
pig’s kidney cells can be also found around them.
When using the probe C9 at co-culturing stained and
non-stained cells the similar results were obtained.
Combined culturing of fibroblast cells labeled with
the JC-1 probe with non-stained SPEV cells has de-
monstrated that there was no transition of the dye from
labeled into non-labeled cells, which enables the moni-
toring of a certain cell line (Fig. 8, A, see inset).
We also have found that at the combined culturing
of JC-1 labeled SPEV and non-stained fibroblasts no
transition of the dye into non-labeled cells was obser-
ved (Fig. 8, B, see inset).
In addition, the duration of survival of the fluores-
cent probes in proliferating culture was studied. It has
been established that luminescence of probes in cells
persisted during the whole period of culture growing
till the next re-plating (4–5 days) and longer (10–
16 days). The luminescence intensity of dyes had a
fading character, decreasing along with the cell fis-
sion. Carbocyanine probes persisted in fissionable cells
throughout 4–8 doublings. In this case localization of
the dyes did not change, and luminescence slightly
decreased.
Discussion. The studied probes of carbocyanine
series were synthesized as analogues of the well-kno-
wn probes. In the papers [11, 12] when investigating an
effect of counterion of the cyanine dyes on photophy-
sical properties, the authors observed the quantum
yield decrease for iodides, if compared with chlorides
or bromides. This is a so-called «heavy atom» effect.
Therefore, we synthesized and used mainly bromides.
We studied the properties of probes C2, C9, C18,
which are the members of alkyl family, differing by the
lengths of alkyl chains to reveal the similarity and
distinction between the functional characteristics and
peculiarities of interaction with the cell cultures. It
should be noted that the number of alkyl groups and the
level of hydrophobicity affect the character of a dye
interactions with intracellular structures [13]. It has
been reported [14–16] that, when using carbocyanine
probes for labeling cells, localization of probes in cells
depends on the applied concentration of dye and the
length of alkyl chains.
At low concentrations (below 10 µM) the probe is
located on the mitochondria membranes, at high
concentrations it reflects the state of endoplasm reti-
culum. According to the Invitrogen-Molecular Probes’
data, the carbocyanine probes with short alkyl chains
(C1–C6) at low dye concentration of 0.5 µM stain the
cell mitochondria, while at concentrations of 5–50 µM
the staining of the sarcoplasmic reticulum is observed.
At the dye concentration used in our experiments the
C18 dye seems to stain the lipid sites of endoplasm
reticulum whereas the C2 and C9 dyes give the in-
formation on the state of mitochondria. It has been
shown that the probe C18 is the most lipophilic, has
different from the other probes of this type localization
in a cell and is characterized by heterogeneous binding
character.
One of the dyes frequently used for the assessment
of the state of mitochondria in cells is the JC-1 probe,
the potential-dependent dye, which specific molecule
interaction with mitochondria depends on the trans-
membrane potential, generated by functioning mito-
chondria. In the diagnostic set MitoPT (Immuno-
chemistry Technologies, USA) the probe JC-1 serves
as a marker of caspase-independent apoptosis due to
tracing the changes in functional state of mitochondria
in cells. Our studies of the synthesized probe JC-1 with
FACS method also testify to adequacy of this appro-
ach. After apoptosis induced by etoposide the shift of
cell luminescence from orange towards green area has
been demonstrated.
We have found that this probe may be successfully
applied for labeling human fibroblasts both at express
diagnostics of a cell state and at long-time stay in cells.
According to the FACS-analysis data, the fluorescence
in orange area is characteristic of the HF cell cultures,
stained with the investigated probe JC-1 that cor-
responds to the luminescence of the mitochondria with
high transmembrane potential. We have shown that the
probes C2 and C9 stain 100 % of cells in the studied
suspension, the stained cell organelles are luminescent
in green area. For the cells stained with the probe C18
the staining of all cells is observed. Their luminescence
is recorded in red area. Our results seem be a ground for
488
GONCHARUK E. I. ET AL.
489
NEWLY SYNTHESIZED CARBOCYANINE FLUORESCENT PROBES
the statement that the application of these probes as
potential-dependent ones is restricted.
The authors of [17] have studied the labeling of bo-
ne marrow cells with the probe of carbocyanine series
CM-DiI (chloromethyl-benzamidodialkylcarbocyani-
ne). CM-DiI is the DiI derivative, hydrophilic proper-
ties of which exceed those of DiIC18(3) (1, 1'-dioc-
tadecyl-3,3,3',3'-tetramethylindocarbocyanine perch-
lorate), that facilitates the preparation of staining solu-
tions for cell suspensions. In the paper [2] the fluores-
cent lipophilic probe CM-DiI was used for monitoring
the bone marrow mesenchymal stromal cells. It has
been shown that CM-DiI is not toxic and does not affect
the cell proliferation, and the intensity of fluorescence
reduces twice after each cell division. This probe
provided the tracing of cells minimum for 30 culturing
days. The dye PKH-26 (Sigma) (structurally identical
to DiIC18(3)), which are also applied for cell labeling
in vivo and in vitro, may be preserved in cells for
21 days.
The carbocyanine probes C2, C9, C18 and JC-1,
obtained by us, at optimal concentrations are not toxic
for the studied cell lines, that is proved by unchanged
morphology and proliferative activity of the studied la-
beled cultures.
The probe, optimal for observation of proliferation
cultures, is the probe C9, which enables the tracing of
mitochondria functional state, due to tight binding and
providing distinct morphological picture of chon-
driome.
We have shown that the studied carbocyanine pro-
bes are preserved in dividing cells during at least four
duplications. It has been found that the carbocyanine
probe JC-1 does not transit from cell to cell at combi-
ned culturing of labeled and non-labeled cells and can
provide the information on a certain cell.
The probes C2, C9, C18 transit from cell to cell.
Spontaneous transcellular transition of the studied pro-
bes under modeled transplantation in vitro does not al-
low recommending investigated probes for tracing the
fate of transplanted cells in the recipient’s organism.
At the same time the long-term survival and invaria-
bility of the probe properties in cultured cells can be a
reason to recommend them for the long-term labeling
of biological objects when necessary (e. g. in low tem-
perature banks, the coding of cell samples during trans-
portation).
The research was carried out under the STCU grant
4358 support.
Authors thank Dr. Dyubko T. S. for her consulting
and fruitful discussion and Timon V. V. for technical
assistance.
О. І. Гон ча рук, І. А. Бо ро вой, О. В. Пав ло вич, Ю. В. Ма люкін,
В. І. Гри щен ко
Но во син те зо вані кар бо цiанінові флу о рес центні зон ди, їхня
ха рак те рис ти ка та по ведінка в проліфе ру ю чих куль ту рах
Ре зю ме
Mета. Дослідити мож ли вості за сто су ван ня кар боціаніно вих
флу о рес цен тних зондів С2, С9, С18 та JC-1 для ха рак те рис ти -
ки куль тур клітин. Ме то ди. Вико рис та но мор фо логічні ме то -
ди, ме тод про точ ної ци тоф лу ори метрії (FACS-аналіз), люмі-
не сцентну мікрос копію. Ре зуль та ти. По ка за но, що дослідже-
ні карбоціанінові зон ди зберіга ють ся в кліти нах, що ділять ся,
про тя гом не менш чотирьох под воєнь. Вста нов ле но, що кар -
боціаніно вий зонд JC-1 не пе ре хо дить із клітини в клітину при
одночасно му куль ти ву ванні міче них і неміче них клітин різних
куль тур. Вис нов ки. Встановлено, що зазначені флу о рес центні
зон ди мож на ви ко ристовувати при дов гот ри вал о му куль ти ву -
ванні клітин них ліній. Для спос те ре жен ня за проліфе ру ю чими
куль турами опти маль ним є застосування зондів С9 і JC-1, що
доз во ляє відслідко ву ва ти функціональ ний стан міто хондрій.
Клю чові сло ва: флу о рес ценція, зон ди, фіброб лас ти лю ди ни,
куль ту ра клітин.
Е. И. Гон ча рук, И. А. Бо ро вой, Е. В. Пав ло вич, Ю. В. Ма лю кин,
В. И. Гри щен ко
Но во син те зи ро ван ные кар бо ци а ни но вые флу о рес цен тные
зон ды, их ха рак те рис ти ка и по ве де ние в про ли фе ри ру ю щих
куль ту рах
Ре зю ме
Цель. Иссле до вать воз мож нос ти при ме не ния кар бо ци а ни но -
вых флу о рес цен тных зон дов С2, С9, С18 и JC-1 для ха рак те -
рис ти ки куль тур кле ток. Ме то ды. Исполь зо ва ны мор фо логи-
чес кие ме то ды, ме тод про точ ной ци тоф лу о ри мет рии (FACS-
ана лиз), лю ми нес цен тная мик рос ко пия. Ре зуль та ты. По ка за -
но, что ис сле ду е мые кар бо ци а ни но вые зон ды со хра ня ют ся в
де ля щих ся клет ках в те че ние не ме нее четырех удво е ний.
Уста нов ле но, что кар бо ци а ни но вый зонд JC-1 не пе ре хо дит из
клет ки в клет ку при со вмес тном куль ти ви ро ва нии ме че ных и
не ме че ных кле ток раз ных куль тур. Вы во ды. Опре де ле но, что
указанные флу о рес цен тные зон ды мож но ис поль зо вать при
дол гов ре мен ном куль ти ви ро ва нии кле точ ных ли ний. Для на -
блю де ния за про ли фе ри ру ю щими куль турами опти маль ным яв -
ля ет ся применение зондов С9 и JC-1, что по зво ляет
от сле жи вать функ ци о нальное со сто я ние ми то хон дрий.
Клю че вые сло ва: флу о рес цен ция, зон ды, фиб роб лас ты че ло -
ве ка, куль ту ра кле ток.
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УДК 57.085.2:577.336
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