[CANCER RESEARCH 47. 6046-6051, November 15, 1987)

Flow Cytometry and Scrape-loading/Dye Transfer as a Rapid Quantitative Measureof Intercellular Communication in Vitro1

Terranee J. Kavanagh,2 George M. Martin, Mohamed H. El-Fouly, James E. Trosko, Chia-Cheng Chang, and

Peter S. RabinovitchDepartment of Pathology- SM-30, University of Washington, Seattle, Washington 98195 [T. J. K., G. M. M., P. S. R.], and Department of Pediatrics and HumanDevelopment, Michigan State University, East Lansing, Michigan 48824 ¡M.H. E-F., J. E. T., C-C. C.]

ABSTRACT

We describe two flow cytometric assays performed on populations ofcells which have been stained with various fluorescent tracer moleculesby the scrape-loading technique. One assay uses a simple one-coloranalysis on a flow cytometer by quantitating the fluorescence intensity ofscrape-loaded lucifer yellow CM (LY) in individual cells. The other assayutilizes a two-color analysis on a cell sorter whereby cells which areinitially loaded (donors) are identified by their uptake of both rhodamineisothiocyanate-dextran an •LY, whereas the recipients of dye transferare identified as having LY only. Agents which have been shown to inhibitintercellular communication in other assays exhibit similar blockingactivity in LY transfer and this is readily quantitated by flow cytometry.The two-color analysis has the added advantage of being able to identifyboth donors and recipients in a highly quantitative manner.

INTRODUCTION

Intercellular communication is an important process in thecontrol of cellular growth and differentiation and tissue home-ostasis (1-4). Aberrations in gap junction-mediated intercellular communication have been identified as a marker for abnormal growth and neoplastic transformation in vitro (5, 6). Inaddition, tumor promoters have been shown to induce alterations in gap junctional communication (4, 7-19) as well ascause disturbances in gap junction morphology both in vitro(20) and in vivo (21).

Because of the postulated importance of cell-cell communication in cellular growth control, many assays have been developed to measure the ability of cells to transfer low molecularweight substances across gap junctions (reviewed in Ref. l).Several methods exist which can yield quantitative data in arapid fashion (electrical coupling; intracellular dye injection),but these require a specialized microinjection apparatus whichcan be difficult to use. Another recently developed methodutilizes fluorescence recovery after photobleaching (18). Although this technique allows for real time analysis of dyetransfer of cells in situ, it utilizes instrumentation that is notyet widely available to most researchers. Other methods tomeasure intercellular communication include metabolic cooperation assays and [-'Hjuridine transfer assays, but these gener

ally require several days or up to a week or more to performthe assay. Quantitation in these assays is often tedious (i.e.,counting silver grains) or requires many tissue culture plates ifthe degree of inhibition of cell-cell communication is only slight(metabolic cooperation assays).

Each of these assays has its advantages, however. For instance, metabolic cooperation assays are useful especially intoxicology studies because the cytotoxicity of test agents can bedetermined simultaneously along with their effects on metaboliccooperation (9, 15, 16). The rapidity and ease with which one

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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by USPHS Grants AC 01751 and CA 09437.2To whom requests for reprints should be addressed, at Department of

Pathology SM-30. University of Washington, Seattle. WA 98195.

can observe cell-cell communication by using the scrape-loading/dye transfer method (7). and the general availability of flowcytometric analysis in most research centers, led us to investigate the possibility of combining scrape-loading/dye transferwith flow cytometry as an additional means of quantitating dyetransfer on a population basis. We describe here two flowcytometric methods, one using a relatively inexpensive non-sorting flow cytometer and the other using a cell sorter which,when combined with the scrape-loading technique, can readilyquantitate the degree of dye transfer between human diploidfibroblasts in vitro.

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 aHDF strain obtained from fetal lung (AC 3542; gift of Dr. G. Raghu,University of Washington Seattle, WA). Cells were grown in modifiedEagle's medium with Earle's balanced salts containing a 50% increase

in vitamins and essential amino acids (except glutamine) and a 100%increase in nonessential amino acids. The medium was further supplemented with 1 mM sodium pyruvate and 10% fetal bovine serum. Cellswere incubated at 37°Cin humidified air containing 5% CO2.

Scrape Loading. The scrape-loading/dye transfer technique has beendescribed in detail (7). In brief, one day prior to an experiment, cellswere 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 toscrape loading. The cells were washed twice with PBS (Gibco, GrandIsland, NY). The cultures were flooded with a PBS solution containing2 mg/ml of LY only (for one-color analyses) or a solution containingLY (2 mg/ml) and RITC-D (2.5 mg/ml) for two-color analyses. Scrapeloading was performed with a "comb" constructed from a 15-mm lineararray of metal needles. The comb was rotated in 360°arcs such that

concentric circles of cells were left on the dish. The dishes were thenincubated for 5 min (except in cases in which rates of transfer werebeing investigated) to allow for equilibration of the dye across gapjunctions. The dye mixture was then aspirated and dishes were washedwith PBS containing EDTA (0.2 mg/ml) and trypsinized. Cells werethen suspended in medium with 3% fetal bovine serum, stored brieflyat 4°Cto prevent LY efflux, and protected from light until flow

cytometric analyses were performed.Flow Cytometric Analyses. Two different types of analyses were

explored for quantitating differences in the degree of dye transfer thathad occurred between cells.

The first method involved a single-color analysis on an arc-lampilluminated flow cytometer (FACS Analyzer; Becton-Dickenson,Mountain View, CA). For these experiments cells were loaded with LYonly. Excitation was at 400-505 nm (LP400 and SP505) and emissionwas detected at 515-545 nm (DF530/30). Fluorescence signals weredisplayed on log scale with simultaneous quantitation of electronic cellvolume, also on log scale. Signals were digitized and stored on a DECPOP-11/23 computer (Digital Equipment Corp., Maynard, MA) togive scattergrams of log volume versus log fluorescence intensity. His-

3The abbreviations used are: LY, lucifer yellow CH; HDF, human diploidfibroblasts; PBS, Dulbecco's phosphate-buffered saline: RITC-D, rhodamine is-othiocyanate dextran: TPA, 12-O-tetradecanoylphorbol-13-acetate.

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tograms of log fluorescence intensity (x-axis) versus cell number (y-axis) were formed from analysis of the scattergram by collapse of thevolume/fluorescence data on the fluorescence axis after clockwise rotation of the axis by 45°.This rotation was necessary to remove volume

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 (Model50H/2151; Ortho Diagnostic Systems, Westwood, MA) equipped withan argon laser (Model 90-6; Coherent, Palo Alto, CA) tuned to 458nm (LY excitation) and a dye laser (Model 599-OEM; Coherent) tunedto 570 nm (RITC-D excitation). Fluorescence emissions of LY andRITC-D were at 575-545 nm and above 590 nm, respectively. Analysiswas gated on the basis of forward scatter to select cells of a nearly

Fig. I. Epifluorescence photomicrographs of scrape-loading and dye transferin HDF monolayers exposed to LY. A, positive LY dye transfer in HDF. Theextent of LY fluorescence indicates efficient gap junction-mediated communication between cells. A noticeable fluorescence gradient can be observed with itshighest intensity in those cells near the line of scraping. B. blockage of LY dyetransfer following pretreatment with a noncytotoxic dose of the potent tumorpromoter TPA (10 ng/ml for I h).

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Fig. 2. Method of analysis of flow cytometry data used to generate histogramsof cell number versus log fluorescence intensity. A, contour plot of cells exposedto 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 analysisis shown for C and D, which display a contour plot of cells scrape loaded withLY and the resulting histogram, respectively.

uniform size for subsequent fluorescence analysis, and thus a volume-based adjustment in fluorescence intensity was not performed.

RESULTS

One-Color Analysis. The principle of scrape-loading/dyetransfer is shown in Fig. 1. LY dye solution was added toconfluent monolayers of HDF and scrape loaded into the cells.As previously shown (7), LY readily transferred into adjacentnonscraped cells (Fig. \A) except when the transfer was blockedby TPA (Fig. 1Ä).

When cultures which have been dye loaded are trypsinizedand analyzed on the flow cytometer, both fluorescence intensityand volume can be simultaneously measured for each cell. Thecells adjacent to the scrape have had their membranes transiently permeabilized to LY. When we compared the fluorescence of cells of different size, there was a direct relationshipbetween cell volume and fluorescent intensity (data not shown),indicating that the cytoplasmic LY concentration had equilibrated with the LY/PBS solution, and the concentration of LY

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Fig. 3. Density dependent transfer of LY after scrape loading as determinedby flow cytometry. Cells (2 X IO5)were plated into either 35- ( ). 60- ( ),or 100- ( ) mm tissue culture dishes. The entire surface of each dish wasscraped such that all dishes contained concentric circles of remaining cells. Thepercentage of LY positive cells was determined by analysis of histograms (upper)obtained as described in Fig. 2. Cells with a fluorescence intensity greater thantwo standard deviations above the mean of the major peak of the histogramgenerated from the low density culture (as determined by a least-squares fittedgaussian curve) were considered LY positive. Data are graphically represented inlower panel.

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was relatively constant within these cells. Since the fluorescencesignals generated by these cells is the sum of their autofluores-cence and LY fluorescence, and since both of these values aredirectly related to volume, we used an analysis which normalized for the volume-related effects. Unlike measurements whichare only indirectly related to cell volume (such as light scatter),the electronic cell volume measurement is ideally suited tocorrectly perform such compensation. Taking the logarithm ofboth volume and fluorescence intensity, distributions with different volume versus fluorescence relationships (i.e., differentslopes on linear plots) have distributions which are at an angleto the axes but are parallel with each other and displaced alongthe Ar-axis.Fig. 2 shows such log-transformed distributions forcells from plates that were not scraped (Fig. 2A) and scraped(Fig. 2C) in the presence of extracellular LY. The volumedependence of such distributions is removed by collapsing thevolume/fluorescence data onto the fluorescence axis after acounterclockwise rotation of the data by 45°to form a histo

gram of cell number (j>-axis) versus fluorescence intensity (x-axis). This procedure is represented in Fig. 2. The rotation ismade at a 45°angle because the gain of the log amplifiers for

volume and fluorescence signals on the FACS analyzer are thesame. This method of analysis allows one to remove volumebias from the fluorescence signals. Histograms of nonscraped

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Fig. 4. Time-dependent transfer of LY after scrape loading as determined byflow cytometry. Cells were exposed to LY, scraped, and trypsinized either immediately (O min), or after 3, 10, and 20 min. Upper, histogrms for 0 ( ),3 ( ), and 20 ( ) min. The number of LY positive cells was determinedas in Fig. 3, fitting the major peak of the histogram generated from O min for LYtransfer. Lower, percentage of LY positive cells versus time allowed for transfer.

and scrape-loaded HDF exposed to LY are shown in Fig. 2, Band D. respectively.

Fig. 3 shows the cell density dependence of LY transfer.Since the transfer of LY from initially loaded cells to adjacentcells is diffusion mediated, the numbers of LY positive cellsincreases as the cell density increases. This is due to a combination of more cells being loaded and greater numbers of cellsreceiving dye by transfer as the probability of cell-cell contactincreases.

The time course for LY transfer in confluent cultures of HDFis shown in Fig. 4. As one can see from Fig. 4, transfer is rapidand nearly complete after 3 min.

The effect of TPA and dieldrin on dye transfer in confluentcultures of HDF is shown in Fig. 5. A dose-dependent block indye transfer is caused by the tumor promoter TPA (Fig. 5/4). Asimilar dose-dependent effect on dye transfer was seen in confluent cultures of HDF cells preexposed to dieldrin (Fig. 5B).Both of these observations are consistent with previously obtained results in metabolic cooperation assays (see "Discussion").

Two-Color Analysis. Since the numbers of LY donor and LYrecipient cells are difficult to determine with the one-coloranalysis (some donor cells may have intermediate fluorescencedue to equilibration with several recipients), it would be preferable to distinguish between LY donor and LY recipient cells.As described previously (7), one way to mark the donor cells isto label them with a second fluorophore which, by virtue of itshigh molecular weight, cannot cross gap junctions.

Cell sorter analysis of LY and RITC-D loaded cells placed insuspension after scrape loading is shown in Fig. 6. Fluorescenceof LY and RITC-D is quantitated independently by excitationfrom two spatially separated laser beams. There are threeregions of interest when this type of analysis is carried out. Theinitially loaded cells appear as a subpopulation of cells that areboth bright red and intermediate to bright green. The recipientsof dye transfer are a subpopulation with intermediate to brightgreen fluorescence and "negative" red fluorescence (actually

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Fig. 5. Effect of TPA and dieldrin on LY transfer in HDF after scrape loadingas determined by flow cytometry. Confluent dishes of HDF were preexposed toeither 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. Thepercentage of LY positive cells was determined as in Fig. 3, fitting the major peakof the histogram of nonscraped controls.

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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 thepresence 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 tocells). The third subpopulation of nonloaded and nonrecipientcells have low green fluorescence and dim red fluorescence.Thus the enumeration of LY recipients and LY donors is easilymade with this type of analysis.

As with the one-color analysis above, this transfer was dependent upon cell density (Figs. 7 and 8). There is an increasingnumber of cells in the LY recipient subpopulation as the densityof the scraped cultures is increased. Fig. 8 also shows that whenTPA is added to these cultures before scrape loading there is adose-dependent reduction in the numbers of LY recipient cells.

As in the single-color analysis with the flow cytometer, thedistribution of cells into these various subpopulations could beaffected by the addition of dieldrin to confluent cultures ofHDF (Fig. 9). A dose-dependent decrease in the numbers ofLY recipients is caused by exposure to dieldrin, indicating ablock in LY dye transfer.

DISCUSSION

The results obtained by combining flow cytometry with thescrape-loading/dye transfer technique demonstrate the utilityof this approach in providing a quantitative measure of dyetransfer. The combination of these two procedures provides anefficient method for assessing the physiology of gap junctional

communication in vitro under a variety of physiological, pharmacological, and toxicological conditions.

The data presented in Figs. 1 and 2 were suggestive of dyetransfer in high density cultures that had been scraped in thepresence of LY. This interpretation is reinforced by experimentswhich showed a cell density dependence (Fig. 3) and a dramaticblock in dye transfer induced by the tumor promoter TPA (Fig.4A). Many studies have shown that TPA can inhibit cell-cellcommunication in vitro (8, 9, 12, 13, 19). It is clear from theresults obtained with both single-color and two-color analysesthat this blocking effect can be rapidly and efficiently quanti-tated with scrape-loading/dye transfer and flow cytometry.

This effect can be shown for compounds other than TPA, asevidenced by the results obtained when HDF were exposed todieldrin. Dieldrin has been shown to be a tumor promoter forliver in vivo (22), a neurotoxin (23), and also an inhibitor ofmetabolic cooperation in Chinese hamster V-79 cells (10) aswell as in human teratocarcinoma cells (11). Again, there wasa clear dose-dependent reduction in the number of cells withLY fluorescence in the single-color analysis (Fig. 4B) and amarked reduction in the number of LY recipients in the two-color analysis (Fig. 8), indicative of a block in dye transfer.

The amount of dye transfer measured by the flow cytometerwould be best expressed quantitatively as the percentage of LYpositive cells less the percentage of initially loaded cells. Since

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100Fig. 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 IO4cells/cm2, respectively), scrape loaded with LY and RITC-D. Cells were plated and scraped asdescribed 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.

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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-coloranalysis (i.e., the cells with intermediate fluorescence intensitycould be either recipients of dye transfer or donors that havetransferred most of the dye originally loaded into them), a morepractical index for one-color studies is the percentage of LYpositive cells. The percentage of LY positive cells can be deter-

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mined by first defining LY negative cells as those that fallwithin a region determined for nonscraped cells. Any cells withfluorescence greater than this region can be considered to beeither recipients or donors of LY, and comparisons are thenmade of the percentage of LY positive cells under differentexperimental conditions. The problem with this analysis is thatone must be careful to scrape the control and test plates equallyso as not to bias the result with different numbers of initiallyloaded cells. This effect can be seen by comparing the histograms in Figs. 3 and 4, where there is an obvious difference inthe numbers of cells with high LY fluorescence.

Because of the attendant problems with quantitating thenumbers of LY donor cells when using a one-color analysis, we

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CELL-CELL COMMUNICATION MEASURED BY FLOW CYTOMETRY

have also demonstrated a two-color analysis on a cell sorterequipped with a dual excitation source. The principles of LYdye transfer do not differ in this analysis from the flow cyto-metric data presented above. However, when the cells are simultaneously exposed to a high molecular weight marker thatcan be loaded into cells but not transferred across gap junctions,they can be identified as initially loaded cells. The numbers ofLY donors (cells with bright red fluorescence) can thus bereadily quantitated. The numbers of LY recipients can be quan-titated equally as well, for they have predominantly greenfluorescence due to LY transfer. Thus, the analysis becomeshighly quantitative and can be expressed as the numbers of LYrecipients (cells with LY only) per LY donor (cells with LY andRITC-D). Although the use of the cell sorter with two-coloranalysis requires more sophisticated instrumentation and tuning, the results are more quantitative than those obtainable witha one-color analysis because of the ability to readily identifyand quantitate these dye donor and recipient cells. An additional benefit of using the sorter is the ability to sort cells fromthe population which remain negative after scrape loading. Thisshould allow for the selection of variants in the population withreduced metabolic cooperation for further study.

We are currently investigating the use of other large molecular weight fluorophores other than RITC-D so that a two-color analysis can be performed on equipment with a singleexcitation source. A combination with potential prospectsmight be a low molecular weight phycobiliprotein together withLY. The phycobiliproteins can be excited at wavelengths closeto those used to excite LY and yet have emission characteristicswhich allow good separation of their signals. Evaluations ofphycobiliprotein subunits and fragments are in progress.

Use of flow cytometry in quantitating dye transfer by thetechniques described here has potential applications in not onlytoxicology and chemical carcinogenesis research (i.e., thescreening of putative tumor promoters and the investigation ofchemically induced perturbations in gap junctions) but also ininvestigating the effect of disturbances in gap junctional communication on embryonic development and differentiation (24).

Furthermore, this approach may have utility in the field ofcancer diagnosis. In a series of experiments designed to determine the communication competence of a host of continuouscell lines, it was found that there was a good correlation betweenthe ability of these cells to undergo cell-cell communicationwith feeder layers of normal cells and their ability to be growtharrested by the normal cells (6, 25). Since abnormalities in cell-cell communication might be an early marker of transformation(5, 6, 21, 25), an exciting possibility would be to use thetechniques described here to assess the cell-cell communicationpotential of premalignant tissues as a prognostic indicator.

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18. Wade, M. H.. Trosko, J. E., and Schindler, M. A fluorescent photobleachingassay of gap-junction-mediated communication between human cells. Science(Wash. DC), 232: 525-528. 1986.

19. Yotti, L. P.. Chang, C. C., and Trosko, J. E. Elimination of metaboliccooperation in Chinese hamster cells by a tumor promoter. Science (Wash.DC), 206: 1089-1091, 1979.

20. Yancey. S. B., Edens, J. E., Trosko, J. E., Chang, C. C., and Revel, J-P.Decreased incidence of gap junctions between Chinese hamster V79 cellsupon exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.Exp. Cell Res., 139: 329-340, 1982.

21. K,ilimi. G. H.. and Sirsat, S. M. The relevance of gap junctions to stage Itumor promotion in mouse epidermis. Carcinogenesis (Lond.), 5: 1671-1677, 1984.

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23. Drewes, C. D., and Vining, E. P. In vivo neurotoxic effects of dieldrin ongiant nerve fibers and escape reflex function in the earthworm Eisenia foetida.Pestic. Biochem. Physiol., 22: 93-103. 1984.

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1987;47:6046-6051. Cancer Res   Terrance J. Kavanagh, George M. Martin, Mohamed H. El-Fouly, et al.  

in VitroQuantitative Measure of Intercellular Communication Flow Cytometry and Scrape-loading/Dye Transfer as a Rapid

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