2006_ flow cytometry analysis of gap junction

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Review Article
Flow Cytometry Analysis of Gap Junction-
Mediated Cell–Cell Communication:
Advantages and Pitfalls
Paula Candida Fonseca,1,2 Oscar Kenji Nihei,3 Wilson Savino,3 David C. Spray,4
and Luiz Anastacio Alves1*
1Laborat�orio de Comunicação Celular, Departamento de Imunologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz,
Rio de Janeiro, Brasil
2 Centro de Ciências da Sa�ude, Instituto de Ciências Biom�edicas, Programa de P�os-Graduação em Ciências Morfol�ogicas,
Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brasil
3Laborat�orio de Pesquisas sobre o Timo, Departamento de Imunologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz,
Rio de Janeiro, Brasil
4Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York
Received 8 June 2005; Accepted 11 October 2005
Background: Since the first morphological description
of the gap junctions use electron microscopy, a consid-
erable number of techniques has been introduced to
evaluate gap junction channel functionality, many of
which use dye transfer techniques, such as dye injec-
tion and fluorescent dye transfer, analyzed by flow cyto-
metry.
Methods: To analyze dye transfer, generally one popula-
tion of cells is incubated with calcein-AM (0.5 lM) for
30 min at 37�C, and the other population was incubated
with the lipophilic dye DiIC18 (3) (10 lM) for 1 h at
37�C; after incubation, these cells were washed five
times with PBS and cocultured for different times, and
then the dye transfer was analyzed by flow cytometry.
Results: In this short overview, we focus on some advan-
tages and disadvantages of flow cytometry as a technique
to investigate gap junction-mediated intercellular commu-
nication (GJIC). In addition, we point out some technical
pitfalls that we have encountered when applying this tech-
nique to study gap junctions in immune system cells.
Conclusions: Analysis of fluorescent dye transfer by flow
cytometry is a useful tool to investigate GJIC. However,
some points must be taken into consideration before using
this methodology, which are discussed herein. q 2006 Inter-
national Society for Analytical Cytology
Key terms: gap junctions; flow cytometry; intercellular
communication; lymphocytes
Gap junctions are intercellular channels that directly
connect adjacent cells, allowing bidirectional passage of
ions, metabolites, and molecules up to 1 kDa in mamma-
lian cells (1,2). The alignment of two compatible hemi-
channels, in the membranes of adjacent cells, forms the
complete gap junction channel. Each hemichannel is a
hexamer of connexin proteins (Cx), composed either by
equal isoforms (homomers) or different ones (heteromers)
(3). Twenty-one different connexin isoforms have been
cloned and identified so far in rodents; connexins are
named according to their MW in kDa (e.g., Cx43 is the 43 kDa
connexin isoform). Each of these connexins confers distinct
properties to the corresponding gap junction channel in
terms of biophysical properties, permeability, ion selectiv-
ity, and compatibility with other connexins (4).
It has been demonstrated that gap junction-mediated
intercellular communication (GJIC) plays multiple roles in
a variety of physiological phenomena, such as early devel-
opmental regulation, cell growth, oncogenic transformation,
hormone secretion, and electrical coupling in the central
nervous system, and in cardiac and smooth muscle tissues
(5–8).
Different strategies have been used to evaluate gap junc-
tion functionality, including metabolic cooperation, radio-
active nucleotide transfer, fluorescent dye microinjection,
scrape-loading, dual whole cell patch-clamp electrophysi-
Contract grant sponsors: FAPERJ, CNPq, and NIH.
*Correspondence to: Luiz Anastácio Alves, Laboratório de Comunicac̨ão
Celular, Departamento de Imunologia, Instituto Oswaldo Cruz, Fundação
Oswaldo Cruz–FIOCRUZ, Av. Brasil, 4365 Manguinhos, 21045-900 Rio de
Janeiro, Brasil. E-mail: alveslaa@ioc.fiocruz.br
Published online 27 April 2006 in Wiley InterScience (www.
interscience.wiley.com).
DOI: 10.1002/cyto.20255
q 2006 International Society for Analytical Cytology Cytometry Part A 69A:487–493 (2006)
ology, standard eletrophysiology, fluorescence recovery af-
ter photobleaching, local activation of a molecular fluores-
cent probe, and the so-called ‘‘parachute assay,’’ in which
cells labeled with a gap junction permeant indicator are
plated atop unlabeled cells (9–14).
In the last few years, an increasing number of publica-
tions have reported the use of flow cytometry as the sole
or complementary technique to detect and quantify GJIC
in different cell preparations (see examples in Table 1).
Eva Guinan and coworkers (26) published the first use
of this technique in gap junction studies, which described
the flow cytometric detection of heterocellular junctional
coupling between peripheral blood lymphocytes and vas-
cular endothelial cells. In this report, one cellular popula-
tion was loaded with the membrane permeable fluores-
cent dye BCECF-AM that is readily retained in the cytosol
after the acetoxymethyl ester is cleaved by intracellular
esterases. The resulting compound has a molecular weight
of 520 Da, and thus is able to pass through gap junctions.
Donor and recipient cells were cocultured for different
times to ascertain dye transfer, and, as control, the MDCK
cell line (which displays low functional coupling) was also
evaluated. After the coculture, the identification of donor
and recipient cells was based on the distinct morphology
of endothelial cell and lymphocytes when forward (size)
and side (granularity) scatter parameters (FSC and SSC,
respectively) were considered.
Following this first description, other investigators
incorporated modifications to this technique, allowing its
utilization to evaluate homocellular junctional communi-
cation. In these studies, two fluorescent dyes have been
applied—a lipophilic one which labels the cell membrane—
generally PKH-26 or DiIC18 (3); and a second fluorochrome
that loads its cytosol (generally BCECF-AM or Calcein-AM),
allowing cytometric identification of donor and recipient
cells even when both are of the same cell type (31–34). The
paradigm for such an experiment is illustrated in Figure 1.
Flow cytometric analysis of functional GJIC provides
several advantages when compared to other techniques,
including analysis of a large cellular population, easy eva-
luation of homocellular and heterocellular communication,
identification of different cellular subpopulations, and very
high sensitivity, which allows detection of subtle differences
in dye coupling. However, this strategy still faces some pit-
falls that may lead tomisinterpretation of data obtained.
In the present overview, we discuss the different param-
eters that may be evaluated when intercellular communi-
cation is analyzed by flow cytometry. In addition, we point
Table 1
Characteristics of Gap Junction-Mediated Intercellular Communication in Different Cell Preparations Evaluated by Flow Cytometry
Intercellular
communication
Donor cell
labeling
Recipient cell
labeling
Gap junction
blockers
Possible technical
pitfalls Reference
T/fiT/ Calcein-AM DiIC18(3) Heptanol Nonspecific calcein uptake 15
IT-76M1fiIT-76M1a Calcein-AM DiIC18(3) Carb./18b-glyc. Nonspecific DiIC18 uptake Figure 3
SSC-13fiSSC-13a Calcein-AM PKH-26 Heptanol Nonspecific PKH-26 uptake 16
PA317afi9La Calcein-AM PKH-26 Not used Nonspecific PKH-26 uptake 17
L/ TfiL/ T Calcein-AM/
DiIC18(3)
Unlabeled 18-a glyc./GAP 27 Nonspecific blocking 18
L/ TfiL/ B Calcein-AM/
DiIC18(3)
Unlabeled 18-a glyc./GAP 27 Nonspecific blocking 18
L/ BfiL/ T Calcein-AM/
DiIC18(3)
Unlabeled 18-a glyc./GAP 27 Nonspecific blocking 18
L/ BfiL/ B Calcein-AM/
DiIC18(3)
Unlabeled 18-a glyc./GAP 27 Nonspecific blocking 26
ROSafiROSa Calcein-AM PKH-26 Not used Nonspecific PKH-26 uptake 19
L87/4afiCD341 cells Calcein-AM Unlabeled Heptanol Impossible to determine 20
M/fiIEC Calcein-AM Unlabeled Heptanol Impossibleto determine 21
HTLV-1 transformed
CD41 T cells fiEC
Calcein-AM Unlabeled 18-a glyc. acid Impossible to determine 22
SGC1afiSGC1a Calcein-AM/
Dialkylcarb dye
Unlabeled Not used Impossible to determine 23
S-17afiS17a Calcein-AM DiIC Carb. — 24
S-17afiLeukemic cells Calcein-AM Unlabeled Carb. Impossible to determine 25
SCfiHBMC Calcein-AM Unlabeled Carb. Impossible to determine 25
PBLfiEC BCECF-AM Unlabeled Not used Impossible to determine 26
ECfi PBL BCECF-AM Unlabeled Not used Impossible to determine 26
HUVECfiPMN Calcein-AM Unlabeled Peptides/18-a glyc. Impossible to determine 27
PMNfiHUVEC Calcein-AM Unlabeled Peptides/18-a glyc. Impossible to determine 27
CardiomyocytesfiEPC Calcein-AM DiL-acLFL PMA Nonspecific DiL-acLDL uptake 28
A 431afiM/ Calcein-AM Unlabeled — — 29
HUVECfiM/ Calcein-AM Unlabeled — — 29
BICR/M1Rk
afiBICR/M1Rk Calcein-AM/DiI Unlabeled — Impossible to determine 30
aCell lines.
T/, thymocytes; L/, lymphocytes; EC, endothelial cells; HTLV-1, human T-cell lymphotropic virus type-1; M/, monocytes; Dialkylcarb.
dye, dialkylcarbocyanine dye; IEC, intestinal epithelial cells; SC, stromal cells; PBL, peripheral blood lymphocytes; HBMC, hematopoietic
bone marrow cells; PMN, polymorphonuclear leukocytes; HUVEC, human endothelial cells; EPC, endothelial progenitor cells; PMA, phor-
bol 12-myristate 13-acetate; 18-a glyc., 18-a glycyrrhetinic acid; Carb., carbenoxolone.
488 FONSECA ET AL.
out some pitfalls that we have observed using this tech-
nique, to evaluate gap junctions between cells of the im-
mune system.
FLOW CYTOMETRY: A USEFUL APPROACH TO
STUDY GAP JUNCTIONAL COMMUNICATION
One major advantage of using flow cytometry to study
modulation of gap junctions is the large number of cells
that can be analyzed simultaneously (at least 104 cells).
Moreover, the analysis accuracy and the sensitivity to dis-
criminate variation in fluorescence intensity have further
advantages, which bias the choice of this assay over
others, especially when applied to measurements of the
effects of chemicals on junctional communication.
By comparison, when we use the dye injection assay, it
is difficult to determine subtle differences in gap junction
coupling as a consequence of treatment.
The high sensitivity of flow cytometry assay of dye
transfer, moreover, allows a precise evaluation of the
amount of transferred dye in recipient cells by analyzing
their mean fluorescence intensity (MFI), using the appro-
priate detector. Several groups have demonstrated modu-
lation of gap junction-mediated cell–cell communication
by flow cytometry. Studying homocellular calcein transfer
by flow cytometry among MMT22 cells (a mouse mam-
mary tumor cell line), Kiang and coworkers (33) observed
an upregulation (twofold) of gap junction-mediated dye
transfer by forskolin treatment (deduced EC50: �10 lM);
with the dye transfer being totally abolished by the gap
junction blocking compound heptanol (3 mM). In addi-
tion, Piechocki and coworkers (23) detected a twofold
increase of gap junction-mediated calcein transfer when
evaluating the effect of the antitumor drug docetaxel on
murine salivary gland carcinoma cells. The same authors
also observed an increase of Cx43 protein after the treat-
ment. This methodology was also used to study the effect
of general anesthetics on GJIC in the P19 cell line (35),
and to study the pathway involved in the trafficking and
assembly of Cx31 into GJIC in transfected HeLa cells trea-
ted with different drugs that interfere with cytoskeleton
filaments (36).
In order to more thoroughly evaluate the advantages of
flow cytometry in the analysis of gap junction modulation,
we have studied a mouse thymic epithelial cell line (TEC),
named IT-76M1 cells, which expresses functional gap
junctions (37). One population of TEC cells (donor) was
loaded with cell permeant calcein-AM and the other popu-
lation (recipient) was labeled with DiIc18 (3). As depicted
in Figure 2, establishment of functional dye transfer
among TEC cells can be demonstrated by the detection of
double positive (DP) DiIc18 (3)
1calcein1 cells after the co-
culture period. We detected an increase (�threefold) in
calcein MFI in the DiIc18 (3)
1calcein1 cells obtained from
8-Br-cAMP-treated cocultures, when compared with con-
trol cells (Fig. 2A). Conversely, treatment with the tumor-
promoting phorbol ester, PMA, had the opposite effect,
reducing the calcein MFI of DiIc18 (3)
1 calcein1 cells in
comparison with control cells. These experiments demon-
strate the sensitivity of this technique to detect both posi-
tive and negative modulations of gap junction-mediated
cell–cell communication.
Another important parameter that can be evaluated by
flow cytometry is the extent of heterocellular communica-
tion, mediated by gap junctions. In the case of cells with
obvious distinct cell morphology, the dye transfer assay
may be performed using only the permeable dye (calcein-
AM or BCECF-AM) (21,26,38). However, if the two cell
populations are indistinguishable by size (FSC) and granu-
larity (SSC) parameters, the strategy of labeling one cell
population to distinguish donor from recipient cells is
necessary (18,23,31,33).
This methodology is also a useful tool to compare the
influence of cell-type and connexin-type on coupling prop-
erties, since changing the donor and recipient cells allows
comparison of dye transfer efficiency by different connexin
isoforms and distinct cell types (19,21,33,34,39,40). For
FIG. 1. Dye transfer protocol. To analyze homocellular dye transfer
between IT-76M1 or between N2A cells, one population of cells was incu-
bated with calcein-AM (0.5 lM) for 30 min at 37�C, and the other popula-
tion was incubated with the lipophilic dye DiIC18 (3) (10 lM; Molecular
Probes, Eugene, OR, USA) for 1 h at 37�C. After incubation, these cells
were washed five times with PBS and enzymatically dissociated using
Trypsin–EDTA for 5 min. The donor cells (calcein1 DiIC2) and the recipi-
ent cells (calcein2DiIC1) were cocultured at 1:1 ratio for different times
(3–6 h) in RPMI 1640 medium with 10% SBF.
489FLOW CYTOMETRY ANALYSIS OF GAP JUNCTION-MEDIATED CELL–CELL COMMUNICATION
example, using flow cytometry, Czyz and coworkers (34)
observed that Cx43-transfected HeLa cells transferred cal-
cein more efficiently than did Cx40-transfected HeLa cells.
In addition, evaluation of dye coupling in cells of the same
type (homospecific) and expressing the same gap junction
protein (homotypic) showed that the speed of calcein
spread through Cx43 channels depended on the cell type.
For instance, the rate of dye transfer was higher in a rat
mammary carcinoma cell (BICR/MIR) than in HeLa cells or
3T3/SV40 cells; all expressing Cx43. Moreover, although
both homotypic–heterospecific (same connexin, different
cell types) or heterotypic–homospecific (different connex-
ins, same cell type) coupling occurred, it became delayed
and less efficient compared to homotypic–homospecific
coupling. Finally, heterotypic–heterospecific coupling
between Cx43-expressing BICR/MIR and Cx40-transfected
HeLa cells could not be detected.
Using flow cytometry, dye transfer methodology, and
dye microinjection of three negatively charged fluorescent
dye, Koval and coworkers (19) studied lucifer yellow, cal-
cein, and hydroxycoumarin carboxylic acid, with different
molecular weights, which showed that heterotypic chan-
nels formed by Cx43 and Cx45 can decrease molecular
permeability of intercellular communication between ROS
cells when compared with homotypic channels formed by
Cx43, suggesting that the expression of one connexin can
alter intercellular communication mediated by another
connexin.
A variety of aspects in cell types may account for the dif-
ferences of the dye transfer efficiency, including different
expression of cell adhesion molecules that could interfere
in avidity of cell interaction; different cell sizes that could
interfere in the amount of the dye spread; differences in
the number of functional gap channels; differencesin con-
nexin expression; and different protein kinases that may
modify coupling, as illustrated in the Figure 2.
Although the differences in dye coupling for different
connexins expressed in the same cell type are generally
interpreted as differences in channel permeability, an im-
portant caveat in such studies is that the number of gap
junction channels must be the same.
Finally, flow cytometry allows the evaluation of multiple
parameters at the same time. In this way, the performance
of dye transfer assay with the concomitant utilization of
different antibodies allows determination of the pheno-
type of the cell subpopulations participating in the pro-
cess. As an example, Hurtado and coworkers (24), using
antibodies against CD11b (Mac1) and CD19 (B220),
detected dye transfer between bone marrow stromal cells
and a myeloid Mac-11 lineage, whereas dye transfer between
bone marrow stromal cells and the B lymphocyte lineage
was absent. Consistent with these data, Paranaguass�u-Braga
and coworkers (25), using antibodies against CD34 and
CD45 subsequent to a calcein transfer assay by flow cyto-
metry, demonstrated that 80% of the more immature
CD341CD451 hematopoietic cells do connect to the stroma
through gap junctions.
NONSPECIFIC DYE TRANSFER
In spite of the advantages discussed earlier, our experi-
ence with this methodology has uncovered several possi-
ble technical pitfalls. As illustrated in Figure 3, a portion of
the donor cells (calcein1 TEC cells) can in some cases
nonspecifically take up red dye from DiIC18 (3) IT-76M,
thereby becoming DP (selected regions). As a conse-
quence, two different populations of DP cells were evident,
one with intermediary and another with high calcein MFI;
however, only the dye transfer between DP cells with inter-
FIG. 2. Modulation of gap junction-dependent dye transfer detected by
flow cytometry. DiIC18 (3)-labeled IT76-M1 cells and calcein-loaded IT76-
M1 cells were either cultured separated and analysed together (coculture
t 5 0) as negative dye transfer control (CT-0 h) or cocultured for 5 h (CT-5
h) as positive control. Treatment of IT76-M1 cocultures with 8-Br-cAMP
increased both the number of coupled cells and the dye transfer rate (A),
whereas phorbol 12-myristate 13-acetate (PMA) treatment had the oppo-
site effect, partially inhibiting the transfer (B). Inhibition of dye transfer in
IT76-M1 cocultures treated with carbenoxolone (Carbe) confirmed that
this effect was mediated by gap junctions (N 5 5).
490 FONSECA ET AL.
mediary calcein MFI could be inhibited by gap junction
blockers. These data, together with the fact that DP cells
presenting high calcein MFI, represent a continuous cell
population with donor cells, differing only by exhibiting an
increasing labeling with DiIC18 (3), which indicate that
these cells represent donor cells that nonspecifically took
DiIC18 (3) from recipient cells. One possible solution to this
problem is to subtract the DP cells with high MFI to calcein.
In order to perform this operation, one would set a gate
around these cells and discount the percentage obtained in
this analysis from that of the DP quadrant.
In addition, Saunders and coworkers (41) studying GJIC
in osteoblastic cell line (ROS17/2.8) using flow cytometry
dye transfer observed a nonspecific DiIC3 uptake by the
receptor cells (unlabeled) from the donor cells (Calcein1
DiIC3
1), which the authors ascribed to be an artifact.
Similar unspecific labeling was observed by Rudkin and
coworkers (16), who studied the effect of retinoids on cell
growth and gap junctional communication in squamous
cell carcinoma. The authors found a significant reduction
in gap junction communication after 24-, 48-, and 96-h of
treatment with retinoids. However, at 48- and 96-h of
treatment, a PKH 261 cell population with calcein fluores-
cence intensity as high as that of the donor population
was evident. This population did not modulate calcein
content after the treatment, differing from the population
with low calcein levels, and appeared even in control con-
ditions, suggesting that these cells represent donor cells
that had taken PKH-26 nonspecifically. This possibly occurs
through unknown mechanisms that permit the membrane
exchange of some hydrophobic or hydrophilic molecules
between cell membranes (42).
In addition, we observed nonspecific uptake of calcein
in homocellular coculture of freshly isolated thymocytes
(Fig. 4) and in the thymic lymphoma EL-4 cell line (15).
Similarly, we observed nonspecific (not inhibitable) cal-
cein uptake by non- and adherent thymocytes cocultured
with calcein-loaded TECs (data not shown). Since such
nonspecific calcein uptake is observed in conditions
under which recipient cells are cocultured with donor
cells, which were previously washed and incubated with
medium alone, to allow the passive release of nonpro-
cessed calcein, such unspecific labeling may be attributed
to a yet unidentified mechanism of active calcein exclu-
sion from donor cells.
A similar phenomenon was reported by Martin and
coworkers (38), when incubating unlabeled monolayers
of intestinal epithelial cells with supernatants collected
from calcein-labeled cells; they observed a dose-depend-
FIG. 3. Detection of unspecific DiIC18 transfer among IT76-M1 cells.
DiIC18 (3)-labeled IT76-M1 cells and calcein-loaded IT76-M1 cells were
cultured separately (A) or cocultured for 6 h (B) as negative and positive
dye transfer controls, respectively. In the positive control (B), we identi-
fied two DP populations, one with low and another with high calcein
MFI. Using carbenoxolone treatment (C), we demonstrated that the for-
mation of the population with high calcein MFI is not inhibited, demon-
strating that DiIC18 (3) transfer to some calcein donor cells might occur
nonspecifically (circle) (N 5 3).
FIG. 4. Detection of nonspecific calcein transfer from TEC to thymo-
cytes. Total thymocytes cocultured atop a monolayer of calcein-loaded
TECs for 3 h (CT-3 h) exhibited calcein uptake when compared with fresh
thymocytes (CT-0 h). The dye transfer from TECs to thymocytes was not
inhibited by carbenoxolone (Carbe) treatment in 3 h of coculture, demon-
strating that it was not mediated by gap junctions (N 5 5).
491FLOW CYTOMETRY ANALYSIS OF GAP JUNCTION-MEDIATED CELL–CELL COMMUNICATION
ent increase in calcein MFI in these cells. The authors uti-
lized the level of nonspecific dye transfer as control to
determine the threshold, where the specific dye transfer
could be detected. Thus, only values 0.5-fold and 2.0-fold
above control calcein MFI were considered significant for
cocultures where donor cells were loaded with 0.5 or 1.0
lM of calcein, respectively.
We also observed dye transfer in N2A cells (Fig. 5), a
neuroblastoma cell line that are gap junction deficient
(43,44); the treatment with carbenexolone did not abolish
the dye transfer observed, indicating that this dye transfer
was mediated by an unknown gap junction-independent
mechanism.
Based on the results mentioned earlier, it is mandatory
to use gap junction inhibitors when evaluating gap junc-
tion-mediated cell–cell communication by flow cytometry.
However, since no specific gap junction blocker is yet
available, whenever possible it is highly recommended to
use more than one inhibitor to verify the role of gap junc-
tions in the process being investigated. Alternatively, it is
advisable to compare primary cells obtained from wild-
type with those from connexin null mice or to compare
transfected cells with communication-deficient parentals.
Finally, since any methodology could produce false-posi-
tive results when not properly performed, it is worthwhile
to use more than one technique to demonstrate gap junc-
tion communication in systems in which these channels
are not fully characterized.
ACKNOWLEDGMENTS
We thank Dr. Ana Cristina Martins de Almeida Nogueira
for critical review of the manuscript.
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