[CANCER RESEARCH 47. 6046-6051, November 15, 1987)
Flow Cytometry and Scrape-loading/Dye Transfer as a Rapid Quantitative Measure
of Intercellular Communication in Vitro1
Terranee J. Kavanagh,2 George M. Martin, Mohamed H. El-Fouly, James E. Trosko, Chia-Cheng Chang, and
Peter S. Rabinovitch
Department of Pathology- SM-30, University of Washington, Seattle, Washington 98195 [T. J. K., G. M. M., P. S. R.], and Department of Pediatrics and Human
Development, Michigan State University, East Lansing, Michigan 48824 ¡M.H. E-F., J. E. T., C-C. C.]
can observe cell-cell communication by using the scrape-load
ing/dye transfer method (7). and the general availability of flow
cytometric analysis in most research centers, led us to investi
gate the possibility of combining scrape-loading/dye transfer
with flow cytometry as an additional means of quantitating dye
transfer on a population basis. We describe here two flow
cytometric methods, one using a relatively inexpensive nonsorting flow cytometer and the other using a cell sorter which,
when combined with the scrape-loading technique, can readily
quantitate the degree of dye transfer between human diploid
fibroblasts in vitro.
ABSTRACT
We describe two flow cytometric assays performed on populations of
cells which have been stained with various fluorescent tracer molecules
by the scrape-loading technique. One assay uses a simple one-color
analysis on a flow cytometer by quantitating the fluorescence intensity of
scrape-loaded lucifer yellow CM (LY) in individual cells. The other assay
utilizes a two-color analysis on a cell sorter whereby cells which are
initially loaded (donors) are identified by their uptake of both rhodamine
isothiocyanate-dextran an •
LY, whereas the recipients of dye transfer
are identified as having LY only. Agents which have been shown to inhibit
intercellular communication in other assays exhibit similar blocking
activity in LY transfer and this is readily quantitated by flow cytometry.
The two-color analysis has the added advantage of being able to identify
both donors and recipients in a highly quantitative manner.
MATERIALS
AND METHODS
Chemicals. LY3 and RITC-D (average molecular weight of 10,000,
neutral charge) were purchased from Molecular Probes, Inc. (Eugene,
OR). TPA and dieldrin were obtained from Sigma Chemical Co. (St.
Louis, MO).
Cells and Cell Culture. The cells used in these experiments were a
HDF strain obtained from fetal lung (AC 3542; gift of Dr. G. Raghu,
University of Washington Seattle, WA). Cells were grown in modified
Eagle's medium with Earle's balanced salts containing a 50% increase
INTRODUCTION
Intercellular communication is an important process in the
control of cellular growth and differentiation and tissue homeostasis (1-4). Aberrations in gap junction-mediated intercellu
lar communication have been identified as a marker for abnor
mal growth and neoplastic transformation in vitro (5, 6). In
addition, tumor promoters have been shown to induce altera
tions in gap junctional communication (4, 7-19) as well as
cause disturbances in gap junction morphology both in vitro
(20) and in vivo (21).
Because of the postulated importance of cell-cell communi
cation in cellular growth control, many assays have been devel
oped to measure the ability of cells to transfer low molecular
weight substances across gap junctions (reviewed in Ref. l).
Several methods exist which can yield quantitative data in a
rapid fashion (electrical coupling; intracellular dye injection),
but these require a specialized microinjection apparatus which
can be difficult to use. Another recently developed method
utilizes fluorescence recovery after photobleaching (18). Al
though this technique allows for real time analysis of dye
transfer of cells in situ, it utilizes instrumentation that is not
yet widely available to most researchers. Other methods to
measure intercellular communication include metabolic coop
eration assays and [-'Hjuridine transfer assays, but these gener
ally require several days or up to a week or more to perform
the assay. Quantitation in these assays is often tedious (i.e.,
counting silver grains) or requires many tissue culture plates if
the degree of inhibition of cell-cell communication is only slight
(metabolic cooperation assays).
Each of these assays has its advantages, however. For in
stance, metabolic cooperation assays are useful especially in
toxicology studies because the cytotoxicity of test agents can be
determined simultaneously along with their effects on metabolic
cooperation (9, 15, 16). The rapidity and ease with which one
in vitamins and essential amino acids (except glutamine) and a 100%
increase in nonessential amino acids. The medium was further supple
mented with 1 mM sodium pyruvate and 10% fetal bovine serum. Cells
were incubated at 37°Cin humidified air containing 5% CO2.
Scrape Loading. The scrape-loading/dye transfer technique has been
described in detail (7). In brief, one day prior to an experiment, cells
were plated into 35-mm tissue culture dishes (2 x IO5 cells per dish).
Test chemicals or vehicle controls were added to dishes 2 h prior to
scrape loading. The cells were washed twice with PBS (Gibco, Grand
Island, NY). The cultures were flooded with a PBS solution containing
2 mg/ml of LY only (for one-color analyses) or a solution containing
LY (2 mg/ml) and RITC-D (2.5 mg/ml) for two-color analyses. Scrape
loading was performed with a "comb" constructed from a 15-mm linear
array of metal needles. The comb was rotated in 360°arcs such that
concentric circles of cells were left on the dish. The dishes were then
incubated for 5 min (except in cases in which rates of transfer were
being investigated) to allow for equilibration of the dye across gap
junctions. The dye mixture was then aspirated and dishes were washed
with PBS containing EDTA (0.2 mg/ml) and trypsinized. Cells were
then suspended in medium with 3% fetal bovine serum, stored briefly
at 4°Cto prevent LY efflux, and protected from light until flow
Received 4/14/87; revised 8/5/87: accepted 8/11/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by USPHS Grants AC 01751 and CA 09437.
2 To whom requests for reprints should be addressed, at Department of
Pathology SM-30. University of Washington, Seattle. WA 98195.
cytometric analyses were performed.
Flow Cytometric Analyses. Two different types of analyses were
explored for quantitating differences in the degree of dye transfer that
had occurred between cells.
The first method involved a single-color analysis on an arc-lamp
illuminated flow cytometer (FACS Analyzer; Becton-Dickenson,
Mountain View, CA). For these experiments cells were loaded with LY
only. Excitation was at 400-505 nm (LP400 and SP505) and emission
was detected at 515-545 nm (DF530/30). Fluorescence signals were
displayed on log scale with simultaneous quantitation of electronic cell
volume, also on log scale. Signals were digitized and stored on a DEC
POP-11/23 computer (Digital Equipment Corp., Maynard, MA) to
give scattergrams of log volume versus log fluorescence intensity. His3The abbreviations used are: LY, lucifer yellow CH; HDF, human diploid
fibroblasts; PBS, Dulbecco's phosphate-buffered saline: RITC-D, rhodamine isothiocyanate dextran: TPA, 12-O-tetradecanoylphorbol-13-acetate.
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tograms of log fluorescence intensity (x-axis) versus cell number (yaxis) were formed from analysis of the scattergram by collapse of the
volume/fluorescence data on the fluorescence axis after clockwise ro
tation of the axis by 45°.This rotation was necessary to remove volume
BY FLOW CYTOMETRY
uniform size for subsequent fluorescence analysis, and thus a volumebased adjustment in fluorescence intensity was not performed.
RESULTS
bias from the fluorescence intensity signal (see Fig. 2).
The second method involved a two-color analysis of the fluorescence
intensity of both LY and RITC-D on an Ortho Cytofluorograf (Model
50H/2151; Ortho Diagnostic Systems, Westwood, MA) equipped with
an argon laser (Model 90-6; Coherent, Palo Alto, CA) tuned to 458
nm (LY excitation) and a dye laser (Model 599-OEM; Coherent) tuned
to 570 nm (RITC-D excitation). Fluorescence emissions of LY and
RITC-D were at 575-545 nm and above 590 nm, respectively. Analysis
was gated on the basis of forward scatter to select cells of a nearly
One-Color Analysis. The principle of scrape-loading/dye
transfer is shown in Fig. 1. LY dye solution was added to
confluent monolayers of HDF and scrape loaded into the cells.
As previously shown (7), LY readily transferred into adjacent
nonscraped cells (Fig. \A) except when the transfer was blocked
by TPA (Fig. 1Ä).
When cultures which have been dye loaded are trypsinized
and analyzed on the flow cytometer, both fluorescence intensity
and volume can be simultaneously measured for each cell. The
cells adjacent to the scrape have had their membranes tran
siently permeabilized to LY. When we compared the fluores
cence of cells of different size, there was a direct relationship
between cell volume and fluorescent intensity (data not shown),
indicating that the cytoplasmic LY concentration had equili
brated with the LY/PBS solution, and the concentration of LY
100
a
S so
U-
0 60
o
LU
LU
CE
40
20
Fig. I. Epifluorescence photomicrographs of scrape-loading and dye transfer
in HDF monolayers exposed to LY. A, positive LY dye transfer in HDF. The
extent of LY fluorescence indicates efficient gap junction-mediated communica
tion between cells. A noticeable fluorescence gradient can be observed with its
highest intensity in those cells near the line of scraping. B. blockage of LY dye
transfer following pretreatment with a noncytotoxic dose of the potent tumor
promoter TPA (10 ng/ml for I h).
0
16
32
48
64
FLUORESCENCE INTENSITY
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FLUORESCENCEINTENSITY
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3248
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FLUORESCENCEINTENSITY
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40
60
80
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11IIOniT,r.FNCE INTENSITY
O
16
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FLUORESCENCE
48
64
INTENSITY
Fig. 2. Method of analysis of flow cytometry data used to generate histograms
of cell number versus log fluorescence intensity. A, contour plot of cells exposed
to LY but not scraped: a, degree of rotation (45*): h, data collapsed onto the axis
perpendicular to this angle of rotation, lì.
the resulting histogram. Similar analysis
is shown for C and D, which display a contour plot of cells scrape loaded with
LY and the resulting histogram, respectively.
Fig. 3. Density dependent transfer of LY after scrape loading as determined
by flow cytometry. Cells (2 X IO5)were plated into either 35- (
). 60- (
),
or 100- (
) mm tissue culture dishes. The entire surface of each dish was
scraped such that all dishes contained concentric circles of remaining cells. The
percentage of LY positive cells was determined by analysis of histograms (upper)
obtained as described in Fig. 2. Cells with a fluorescence intensity greater than
two standard deviations above the mean of the major peak of the histogram
generated from the low density culture (as determined by a least-squares fitted
gaussian curve) were considered LY positive. Data are graphically represented in
lower panel.
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CELL-CELL
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was relatively constant within these cells. Since the fluorescence
signals generated by these cells is the sum of their autofluorescence and LY fluorescence, and since both of these values are
directly related to volume, we used an analysis which normal
ized for the volume-related effects. Unlike measurements which
are only indirectly related to cell volume (such as light scatter),
the electronic cell volume measurement is ideally suited to
correctly perform such compensation. Taking the logarithm of
both volume and fluorescence intensity, distributions with dif
ferent volume versus fluorescence relationships (i.e., different
slopes on linear plots) have distributions which are at an angle
to the axes but are parallel with each other and displaced along
the Ar-axis.Fig. 2 shows such log-transformed distributions for
cells from plates that were not scraped (Fig. 2A) and scraped
(Fig. 2C) in the presence of extracellular LY. The volume
dependence of such distributions is removed by collapsing the
volume/fluorescence data onto the fluorescence axis after a
counterclockwise rotation of the data by 45°to form a histo
gram of cell number (j>-axis) versus fluorescence intensity (xaxis). This procedure is represented in Fig. 2. The rotation is
made at a 45°angle because the gain of the log amplifiers for
volume and fluorescence signals on the FACS analyzer are the
same. This method of analysis allows one to remove volume
bias from the fluorescence signals. Histograms of nonscraped
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and scrape-loaded HDF exposed to LY are shown in Fig. 2, B
and D. respectively.
Fig. 3 shows the cell density dependence of LY transfer.
Since the transfer of LY from initially loaded cells to adjacent
cells is diffusion mediated, the numbers of LY positive cells
increases as the cell density increases. This is due to a combi
nation of more cells being loaded and greater numbers of cells
receiving dye by transfer as the probability of cell-cell contact
increases.
The time course for LY transfer in confluent cultures of HDF
is shown in Fig. 4. As one can see from Fig. 4, transfer is rapid
and nearly complete after 3 min.
The effect of TPA and dieldrin on dye transfer in confluent
cultures of HDF is shown in Fig. 5. A dose-dependent block in
dye transfer is caused by the tumor promoter TPA (Fig. 5/4). A
similar dose-dependent effect on dye transfer was seen in con
fluent cultures of HDF cells preexposed to dieldrin (Fig. 5B).
Both of these observations are consistent with previously ob
tained results in metabolic cooperation assays (see "Discus
sion").
Two-Color Analysis. Since the numbers of LY donor and LY
recipient cells are difficult to determine with the one-color
analysis (some donor cells may have intermediate fluorescence
due to equilibration with several recipients), it would be pref
erable to distinguish between LY donor and LY recipient cells.
As described previously (7), one way to mark the donor cells is
to label them with a second fluorophore which, by virtue of its
high molecular weight, cannot cross gap junctions.
Cell sorter analysis of LY and RITC-D loaded cells placed in
suspension after scrape loading is shown in Fig. 6. Fluorescence
of LY and RITC-D is quantitated independently by excitation
from two spatially separated laser beams. There are three
regions of interest when this type of analysis is carried out. The
initially loaded cells appear as a subpopulation of cells that are
both bright red and intermediate to bright green. The recipients
of dye transfer are a subpopulation with intermediate to bright
green fluorescence and "negative" red fluorescence (actually
''V
100
80
60
40
20
0 0.0
0ii
16
BY FLOW CYTOMETRY
32
48
Il
64
FLUORESCENCE INTENSITY
50
I
1.0
CONCENTRATION
40
LU
o
0.5
•
5.0
(ng/ml)
30
«3
100
o.
20
S 80
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5 10
È20
10
¿ 0
20
LY TRANSFER TIME (MIN)
0.0
1.0
I
I
5.0
10.0
CONCENTRATION
Fig. 4. Time-dependent transfer of LY after scrape loading as determined by
flow cytometry. Cells were exposed to LY, scraped, and trypsinized either im
mediately (O min), or after 3, 10, and 20 min. Upper, histogrms for 0 (
),
3(
), and 20 (
) min. The number of LY positive cells was determined
as in Fig. 3, fitting the major peak of the histogram generated from O min for LY
transfer. Lower, percentage of LY positive cells versus time allowed for transfer.
(ug/ml)
Fig. 5. Effect of TPA and dieldrin on LY transfer in HDF after scrape loading
as determined by flow cytometry. Confluent dishes of HDF were preexposed to
either 0.0, 0.5, 1.0, or 5.0 ng/ml TPA (upper); or preexposed to either 0.0, 1.0,
5.0, or 10.0 fig/ml dieldrin (lower) 2 h before being scrape loaded with LY. The
percentage of LY positive cells was determined as in Fig. 3, fitting the major peak
of the histogram of nonscraped controls.
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100
Fig. 6. Isometric displays of HDF exposed to LY (green fluorescence) and RITC-D (red fluorescence) and analyzed by dual laser excitation on a cell sorter. I.
nonscraped cells exposed to LY and RITC-D; B, cells scraped in the presence of LY only; C, cells scraped in the presence of RITC-D only; D, cells scraped in the
presence of both LY and RITC-D. d, RITC-D positive donors; r, LY positive recipients; n, nonlabeled cells.
dimly fluorescent due to nonspecific binding to RITC-D to
cells). The third subpopulation of nonloaded and nonrecipient
cells have low green fluorescence and dim red fluorescence.
Thus the enumeration of LY recipients and LY donors is easily
made with this type of analysis.
As with the one-color analysis above, this transfer was de
pendent upon cell density (Figs. 7 and 8). There is an increasing
number of cells in the LY recipient subpopulation as the density
of the scraped cultures is increased. Fig. 8 also shows that when
TPA is added to these cultures before scrape loading there is a
dose-dependent reduction in the numbers of LY recipient cells.
As in the single-color analysis with the flow cytometer, the
distribution of cells into these various subpopulations could be
affected by the addition of dieldrin to confluent cultures of
HDF (Fig. 9). A dose-dependent decrease in the numbers of
LY recipients is caused by exposure to dieldrin, indicating a
block in LY dye transfer.
DISCUSSION
The results obtained by combining flow cytometry with the
scrape-loading/dye transfer technique demonstrate the utility
of this approach in providing a quantitative measure of dye
transfer. The combination of these two procedures provides an
efficient method for assessing the physiology of gap junctional
communication in vitro under a variety of physiological, phar
macological, and toxicological conditions.
The data presented in Figs. 1 and 2 were suggestive of dye
transfer in high density cultures that had been scraped in the
presence of LY. This interpretation is reinforced by experiments
which showed a cell density dependence (Fig. 3) and a dramatic
block in dye transfer induced by the tumor promoter TPA (Fig.
4A). Many studies have shown that TPA can inhibit cell-cell
communication in vitro (8, 9, 12, 13, 19). It is clear from the
results obtained with both single-color and two-color analyses
that this blocking effect can be rapidly and efficiently quantitated with scrape-loading/dye transfer and flow cytometry.
This effect can be shown for compounds other than TPA, as
evidenced by the results obtained when HDF were exposed to
dieldrin. Dieldrin has been shown to be a tumor promoter for
liver in vivo (22), a neurotoxin (23), and also an inhibitor of
metabolic cooperation in Chinese hamster V-79 cells (10) as
well as in human teratocarcinoma cells (11). Again, there was
a clear dose-dependent reduction in the number of cells with
LY fluorescence in the single-color analysis (Fig. 4B) and a
marked reduction in the number of LY recipients in the twocolor analysis (Fig. 8), indicative of a block in dye transfer.
The amount of dye transfer measured by the flow cytometer
would be best expressed quantitatively as the percentage of LY
positive cells less the percentage of initially loaded cells. Since
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100
100
Fig. 7. Isometric displays showing effect of density and TPA on LY dye transfer in HDF as determined by dual laser analysis performed on a cell sorter. A, B, and
C, cells at low, medium, and high densities (0.25, 0.7, and 2.1 x IO4 cells/cm2, respectively), scrape loaded with LY and RITC-D. Cells were plated and scraped as
described in Fig. 3. D, effect of a 2-h preexposure to 10 ng/ml TPA on the transfer of LY in scrape-loaded high density HDF.
CO
5
_l
uu
O
4
CH MEDIUM DENSITY
LOW DENSITY
O
3
er
05
_j
oÃü
o 4
+
Q
o 3
•I HIGH DENSITY
a
2
Ul
2
0.0
1.0
2.5
5.0
10.0
DIELDRIN(ug/ml)
1
Fig. 9. Effect of dieldrin on LY dye transfer in HDF. The ratio of LY positive
cells to RITC-D positive cells is shown as a function of preexposure to dieldrin
at 0, 1.0, 2.5, 5.0, or 10.0 ng/ml for 2 h.
O
0.0
0.1
1.0
10.0
TPA (ng/ml)
Fig. 8. Effect of density and TPA on LY dye transfer in HDF. The ratio of
LY' positive cells (dye transfer recipients) to RITC-D positive cells (dye transfer
donors) is shown as a function of both cell density and preexposure to TPA at 0,
O.I, 1.0, or 10.0 ng/ml for 2 h.
this last quantity is difficult to determine with a one-color
analysis (i.e., the cells with intermediate fluorescence intensity
could be either recipients of dye transfer or donors that have
transferred most of the dye originally loaded into them), a more
practical index for one-color studies is the percentage of LY
positive cells. The percentage of LY positive cells can be deter-
mined by first defining LY negative cells as those that fall
within a region determined for nonscraped cells. Any cells with
fluorescence greater than this region can be considered to be
either recipients or donors of LY, and comparisons are then
made of the percentage of LY positive cells under different
experimental conditions. The problem with this analysis is that
one must be careful to scrape the control and test plates equally
so as not to bias the result with different numbers of initially
loaded cells. This effect can be seen by comparing the histo
grams in Figs. 3 and 4, where there is an obvious difference in
the numbers of cells with high LY fluorescence.
Because of the attendant problems with quantitating the
numbers of LY donor cells when using a one-color analysis, we
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have also demonstrated a two-color analysis on a cell sorter
equipped with a dual excitation source. The principles of LY
dye transfer do not differ in this analysis from the flow cytometric data presented above. However, when the cells are si
multaneously exposed to a high molecular weight marker that
can be loaded into cells but not transferred across gap junctions,
they can be identified as initially loaded cells. The numbers of
LY donors (cells with bright red fluorescence) can thus be
readily quantitated. The numbers of LY recipients can be quantitated equally as well, for they have predominantly green
fluorescence due to LY transfer. Thus, the analysis becomes
highly quantitative and can be expressed as the numbers of LY
recipients (cells with LY only) per LY donor (cells with LY and
RITC-D). Although the use of the cell sorter with two-color
analysis requires more sophisticated instrumentation and tun
ing, the results are more quantitative than those obtainable with
a one-color analysis because of the ability to readily identify
and quantitate these dye donor and recipient cells. An addi
tional benefit of using the sorter is the ability to sort cells from
the population which remain negative after scrape loading. This
should allow for the selection of variants in the population with
reduced metabolic cooperation for further study.
We are currently investigating the use of other large molec
ular weight fluorophores other than RITC-D so that a twocolor analysis can be performed on equipment with a single
excitation source. A combination with potential prospects
might be a low molecular weight phycobiliprotein together with
LY. The phycobiliproteins can be excited at wavelengths close
to those used to excite LY and yet have emission characteristics
which allow good separation of their signals. Evaluations of
phycobiliprotein subunits and fragments are in progress.
Use of flow cytometry in quantitating dye transfer by the
techniques described here has potential applications in not only
toxicology and chemical carcinogenesis research (i.e., the
screening of putative tumor promoters and the investigation of
chemically induced perturbations in gap junctions) but also in
investigating the effect of disturbances in gap junctional com
munication on embryonic development and differentiation (24).
Furthermore, this approach may have utility in the field of
cancer diagnosis. In a series of experiments designed to deter
mine the communication competence of a host of continuous
cell lines, it was found that there was a good correlation between
the ability of these cells to undergo cell-cell communication
with feeder layers of normal cells and their ability to be growth
arrested by the normal cells (6, 25). Since abnormalities in cellcell communication might be an early marker of transformation
(5, 6, 21, 25), an exciting possibility would be to use the
techniques described here to assess the cell-cell communication
potential of premalignant tissues as a prognostic indicator.
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Flow Cytometry and Scrape-loading/Dye Transfer as a Rapid
Quantitative Measure of Intercellular Communication in Vitro
Terrance J. Kavanagh, George M. Martin, Mohamed H. El-Fouly, et al.
Cancer Res 1987;47:6046-6051.
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