Journal of Histochemistry and Cytochemistry, Vol. 49, 147-154, February 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Spectral Imaging of MC540 During Murine and Human Colon Carcinoma Cell Differentiation

Galit Sibonia, Chana Rothmanna, Benjamin Ehrenbergb, and Zvi Malika
a Microscopy Unit, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
b Physics Department, Bar-Ilan University, Ramat-Gan, Israel

Correspondence to: Zvi Malik, Microscopy Unit, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail: malikz@mail.biu.ac.il


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G1-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147–153, 2001)

Key Words: colon carcinoma, localization, merocyanine, spectral imaging


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

THE FLUORESCENT DYE MEROCYANINE 540 (MC540) is a negatively charged heterocyclic chromophore with a molecular weight of 570 Daltons, which binds preferentially to biological membranes and liposomes (Fiedorowicz et al. 1993 ; Arias et al. 1994 ; Onganer and Quitevis 1994 ; Langner and Hui 1999 ). The extent of binding, and consequently the intensity of fluorescence, is affected by the membrane electric potential and membrane packing (Williamson et al. 1983 ; McEvoy et al. 1998 ). Because of these unique properties, MC540 is used as a membrane probe in the analysis of the metabolic states of both normal and cancer cells (Easton et al. 1978 ; Sieber 1987 ; Anderson et al. 1993 ; Chen et al. 1997 ).

Several parameters affect the spectra, and especially the fluorescence intensity, of MC540. In aqueous solutions and in lipid environments, the dye undergoes dimerization, which is expressed in its spectra (Williamson et al. 1983 ; Singth et al. 1991 ; Yu and Hui 1992 ). Changes in the absorption wavelength of lipid membrane components reflect the shift in equilibrium between monomers and dimers and the reorientation of MC540 molecules near or at the membrane surface. In the presence of a lipid phase, there is strong partitioning of the dye into the lipid membrane. This is accompanied by monomerization (red-shifted absorption bands) and enhanced fluorescence intensity. The emission spectra depend on cholesterol concentration and phospholipid symmetry (Williamson et al. 1983 ; Krieg 1992 ; Onganer and Quitevis 1994 ; Lagerberg et al. 1995 ). MC540 interacts with bilayers with widely spaced lipids (Langner and Hui 1999 ). Therefore, the tendency of malignant cells to bind more dye than normal cells is due to increased membrane fluidity (Onganer and Quitevis 1994 ). Cells whose cholesterol content has been increased are less susceptible to MC540 (Gaffney et al. 1991 ). Therefore, MC540 can assist in monitoring structural changes in the phospholipid bilayer during proliferation and differentiation (Pyatt et al. 1999 ).

In addition to the monomer–dimer equilibrium and aqueous–membrane-bound dye equilibrium, fluorescence intensity is also influenced by the membrane electric potential. In some previous studies, MC540 has been used as a probe for measuring changes in the membrane potential of liposomes (Williamson et al. 1983 ; Ehrenberg and Pevzner 1993 ). These changes depend on two major components of the electric field, the cross membrane potential and the surface potential, and were expressed by a slight alteration in the absorption and fluorescence spectra (Sieber 1987 ; Singth et al. 1991 ; Kaschny and Goni 1992 ; Ehrenberg and Pevzner 1993 ). The first component, also known as the Nernst potential, arises from differences in ion concentrations between the two sides of the membrane, and the second component arises from charged groups at the membrane–water interface.

MC540 has been used in photodynamic therapy of medulloblastomatous and gliomatous brain tumors (O'Brien et al. 1991 ; Whelan et al. 1992 ; Lin and Girotti 1993 ) and for the elimination of tumor cells from autologous bone marrow grafts (Anderson et al. 1993 ; Lin and Girotti 1993 ; Kubo and Sieber 1997 ) and the eradication of enveloped viruses (O'Brien et al. 1992 ; Sieber et al. 1992 ; Lin and Girotti 1993 ). Viruses such as human Herpes simplex virus Type I, human cytomegalovirus, human T-cell leukemia/lymphoma virus Type I, human immunodeficiency virus Type I, Sindbis virus, and Friend erythroleukemia virus are all inactivated in the presence of MC540 (Franck and Schneider 1992 ; Sieber et al. 1992 ; O'Brien et al. 1991 , O'Brien et al. 1992 ). The primary targets of photosensitized MC540 appear to be cell membranes (Singth et al. 1991 ).

The purpose of the present study was to characterize the MC540-activated cell fluorescence of murine CT26 and human HT29 colon carcinoma cells using multipixel spectroscopy and imaging. Spectral changes were correlated with the differentiation state of the cells induced by sodium butyrate as well as with the physiological changes induced by acidic conditions. Spectral mapping functions enabled the detection of specific pixel-by-pixel spectral changes at the subcellular level of a single cell.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Cell Culture
CT26 murine colon carcinoma cells and HT29 human colon carcinoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and antibiotics on tissue culture plates (Nunc; Naperville, IL) in a humidified atmosphere with 5% CO2 at 37C. For transfer, cells were recultured twice a week after detachment using trypsin–EDTA.

Cell Differentiation
The cells were grown on tissue culture plates either with 2.5 mM sodium butyrate or without it for 24 hr or 72 hr. Sodium butyrate was washed out with PBS (-Ca+2/Mg+2) before addition of the nucleic acid stain propidium iodide (PI) in the dark for 15 min. Each solution was filtered and measured by flow cytometry.

MC540 Staining
A stock solution of 1 mM MC540 (Aldrich; Madison, WI) in methanol was prepared. The dye was added to the cells at a final concentration of 10 µM for 1 hr in serum-depleted medium to eliminate the effect of serum elements on the fluorescence intensity.

Fluorescence Measurements
MC540 was diluted in PBS (pH 5.0, 7.2) to a final concentration of 10 µM and the fluorescence intensity was measured by using a spectrofluorometer (Perkin Elmer LS50B; Norwalk, CT) immediately or after 2 hr of incubation with 2.5 mM Triton X-100.

Fluorescence of Liposome-bound MC540
Concentrations of 15 µg/ml and 30 µg/ml liposomes were prepared at pH 5.0 and pH 7.2 and added to PBS (at each pH) with 0.625 µM of MC540 at different concentrations. The solutions were measured by spectrofluorometry (excitation at 495 nm).

Spectral Imaging
CT26 and HT29 cells were grown on glass slides in a buffer bath (pH equal to that of the treatment) and stained with MC540. Spectral image analysis of the stained cells was performed using the SpectraCube SD-200 (Applied Spectral Imaging; Migdal HaEmek, Israel) combined with an AX70 Olympus microscope equipped with a high-pressure mercury lamp for excitation and a set of filters for blue-violet excitation (bandpass 420–480 nm), a dichroic mirror (500 nm), and a cut-on red emission barrier filter (580 nm), and for green excitation (bandpass 545–580 nm), a dichroic mirror (600 nm), and a cut-on red emission barrier filter (610 nm). The SpectraCube system consists of an interferometer and a CCD camera. During a measurement (20 sec), each pixel of the CCD (512 x 512) collects the interferogram, which is then Fourier-transformed to give the spectrum (Malik et al. 1996 ; Rothmann et al. 1998 , Rothmann et al. 1999 ). As a result, spectral imaging acquires a so-called cube whose appellate signifies the two spatial dimensions of a flat sample (x and y), and the third spectrum dimension representing light intensity at any wavelength. The calculated pixel size in a spectral image is 0.04 µm2. The spectral resolution (FWHM, full width at half maximum) is 5 nm at 400 nm (12 nm at 600 nm) and the spectral range (more than 5% response) is 400–1000 nm (Garini et al. 1996 ). The cells were inspected for less than 1 min at a time to avoid bleaching.

Flow Cytometry
CT26 and HT29 cells grown on tissue culture plates were incubated with or without sodium butyrate for 24 hr. The cells were stained with 10 µM MC540 for 1 hr. The cells were washed with PBS (-Ca+2/Mg+2) and scraped off with a rubber policeman. After 5 min of centrifugation at 1100 rpm, the medium was washed out and 0.5 ml of PBS (-Ca+2/Mg+2) was added. The solution was filtered and measured by Fluorescence Activated Cell Sorter (Becton–Dickinson FACS Calibur; Mountain View, CA), with 10,000 cells measured for each sample.

Alkaline Phosphatase (ALP)
CT26 and HT29 cells were grown on tissue culture plates and incubated with 2.5 mM sodium butyrate for 24 and 72 hr. The cells were washed with PBS (-Ca+2/Mg+2) and peeled off with a rubber policeman. After 5 minutes of centrifugation at 1100 rpm, the medium was washed out and 300 µl of PBS (-Ca+2/Mg+2) was added. A total of 100 µl from those cells was added to a test tube filled with 0.5 ml of ALP substrate, p-nitrophenyl phosphate (Sigma 104 phosphatase substrate kit; Sigma Diagnostics, St Louis, MO) at 37C for 15 min. The optical density (OD) of the solutions was measured by spectrophotometer (Novaspec II, Biochrom) at 410 nm. Protein concentration was calculated according to the standard Bradford method (Bradford 1976 ).


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The cellular staining pattern of MC540 in murine CT26 and human HT29 colon carcinoma cells was studied using multipixel spectral analysis. It is well established that the fluorescence intensity of fluorophores is affected by the polarity of their microenvironment (Kaschny and Goni 1992 ; Malik et al. 1997 ) and that their absorbance depends on the ratio between lipophilic and hydrophilic media (Ehrenberg and Pevzner 1993 ). Spectral measurements revealed maximal light absorbance of MC540 at 480 nm in aqueous environments and at 570 nm in lipophilic media and membranes (Fig 1). Therefore, two excitation ranges were set for MC540-stained cells, one at 420–480 nm and the other at 545–580 nm. Measurements were carried out in both physiological and acidic pH media.



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Figure 1. Spectral measurements of the light absorbance of MC540 (6 µM) in distinct environmental conditions: (solid line) in aqueous environment, (dotted line) in lipophilic media.

Excitation of MC540-stained CT26 cells at 420–480 nm, pH 7.2, revealed two cell populations displaying distinct staining patterns (Fig 2A and Fig 2B). In the first pattern, a large number of fluorescent vesicles appeared in the cytoplasm while the nucleus and the plasma membrane remained unstained (Fig 2A). In the second pattern, the cytoplasm was lightly stained and only a small number of vesicles were observed (Fig 2B). Excitation at 545–580 nm, pH 7.2, revealed the same localization patterns as in Fig 2A and Fig 2B (data not shown).



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Figure 2. Localization of MC540 in CT26 and HT29 cells by spectral imaging. The cells were incubated with 10 µM MC540 for 1 hr and analyzed by spectral imaging. Two localization patterns were obtained for MC540 in CT26 cells excited at 420–480 nm at pH 7.2 (A,B) and at pH 6.7 (C,E). Localization of MC540 in CT26 cells excited at 545–580 nm at pH 6.7 (D) and HT29 cells excited at 420–480 nm at pH 6.7 (F). Bar = 6 µm.

MC540-stained CT26 cells at pH 6.7 exhibited three distinct localization patterns (Fig 2C–2E). In cells excited at 420–480 nm, two populations of cells were detected (Fig 2C and Fig 2E). In the first population, MC540 fluorescence was seen in the nuclear membrane, cytoplasmic vesicles, and nucleoli (Fig 2C), whereas excitation of these same cells at 545–580 nm revealed only minor fluorescence in the nucleoli (Fig 2D). In the second population, high fluorescence intensity was detected in the plasma membrane and lower fluorescence intensity in the cytoplasm and nucleoli (Fig 2E). Similarly, in HT29 cells, nucleoli fluorescence was detected only in cells incubated in pH 6.7 medium that were excited at 420–480 nm (Fig 2F). Fluorescence was also detected in the nuclear membrane and in cytoplasmic vesicles. Therefore, nucleoli fluorescence could be observed only by excitation at 420–480 nm under acidic conditions.

Spectra sampled from different subcellular sites of CT26 and HT29 cells displayed the fluorescence spectra shown in Fig 3. In both types of cells incubated at pH 7.2, excitation at 420–480 nm induced fluorescence in the range of 525–600 nm, with maximal intensity at 580 nm in the cytoplasm and at 600 nm in cytoplasmic vesicles (Fig 3A). For cells incubated at pH 6.7, which exhibited fluorescence in the nuclear membrane and nucleoli, excitation at 420–480 nm revealed fluorescence in the range of 550–700 nm, with a maximum at 600 nm (Fig 3B). High fluorescence was detected in the nucleoli and lower fluorescence in the nuclear membrane. In contrast, excitation of the cells at 545–580 nm revealed fluorescence in the range of 600–750 nm, with a maximum at 650 nm. The fluorescence was strong in the nuclear membrane and weaker in the nucleoli.



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Figure 3. Spectral analysis of MC540 in distinct subcellular domains. CT26 and HT29 cells were incubated with 10 µM MC540 for 1 hr at pH 7.2. (A) Cells were excited at 420–480 nm (emission filter >515 nm) and the fluorescence spectra were sampled from the (square) cytoplasm and (solid line) cytoplasmic vesicles. The same spectra were obtained at pH 6.7. (B) Spectra sampled from the nucleolus (open circle) and nuclear membrane (open square) of cells excited at 420–480 nm (emission filter >515 nm) and from the nucleolus (solid circle) and nuclear membrane (solid square) of cells excited at 545–580 nm (emission filter >610 nm).

The spectral pattern of MC540 fluorescence was also studied in differentiating colon carcinoma cells. Differentiation of the murine CT26 and human HT29 cells was induced by sodium butyrate. Cell cycle arrest was detected after 24 hr, at which time 53.3% of CT26 cells and 85.5% of HT29 cells were in the G1-phase (Table 1). In addition, the activity of the specific colon differentiation marker ALP increased markedly with time (Fig 4). After 72 hr the activity of ALP increased by 3.1-fold in CT26 cells and by 4.8-fold in HT29 cells in comparison to the control values. Differentiated CT26 cells (after 72 hr of treatment with sodium butyrate) incubated at pH 6.7 and excited at 420–480 nm showed two populations of cells representing distinct staining patterns. One cell population (Fig 5A) exhibited large fluorescent vesicles in the cytoplasm and staining of the nuclear envelope, whereas in the other (Fig 5B) small vesicles were dispersed in the cytoplasm. Similarly, differentiated HT29 cells incubated under the same conditions revealed significant cytoplasmic fluorescence (Fig 5C). However, no fluorescent nucleoli were detected in the CT26 or HT29 cells treated with sodium butyrate.



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Figure 4. Alkaline phosphatase activity as a function of cell differentiation. CT26 and HT29 were treated with 2.5 mM sodium butyrate to induce differentiation. ALP activity was determined 24 and 72 hr after treatment.



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Figure 5. Localization of MC540 in differentiated CT26 and HT29 cells using spectral imaging. The cells were treated with 2.5 mM sodium butyrate to induce differentiation, incubated with 10 µM MC540 for 1 hr, and analyzed by spectral imaging. Two localization patterns were obtained in CT26 cells (A,B) and one in HT29 cells (C) excited at 420–480 nm at pH 6.7. Bar = 5.5 µm.


 
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Table 1. Cell cycle analysis: percentage of cells in G1

We used flow cytometry to analyze MC540-stained cell populations according to their fluorescence intensity. Acidic conditions are known to induce membrane depolarization which, in turn, increases MC540 binding (Chung et al. 1998 ; Lagerberg et al. 1998 ). Cell sorting analysis by flow cytometry revealed higher fluorescence intensity in cells incubated at pH 5.0 and pH 6.7 than for those incubated at pH 7.2 for both CT26 (Fig 6A) and HT29 cells (Fig 6C). At pH 7.2, two cell populations were observed for undifferentiated CT26 and HT29 cells, but at pH 5.0 only a single population of cells of each type was observed. After induction of differentiation of the cells grown at pH 5.0, the single population of undifferentiated CT26 cells incubated at pH 5.0 diverged into two less fluorescent populations (Fig 6B), while the HT29 cells remained a single population characterized by somewhat reduced fluorescence (Fig 6D).



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Figure 6. Cell sorting analysis of CT26 and HT29 cells. The cells were treated with 10 µM MC540 for 1 hr at pH 5.0 (dotted line) and at pH 7.2 (line). Both undifferentiated (A) CT26 and (C) HT29 cells and differentiated (B) CT26 and (D) HT29 cells were analyzed.

In all treatments, the fluorescence intensity of both CT26 and HT29 cells was higher at pH 5.0 than at pH 7.2. The factors responsible for this phenomenon were investigated in an extracellular system in the presence of either liposomes or Triton. Spectral measurements of MC540 with liposomes showed an increase in the fluorescence intensity in comparison to MC540 alone, but the fluorescence intensity at pH 7.2 remained higher than at pH 5.0 (Fig 7A). However, by dissolving the MC540 aggregates in Triton X-100, the pH effect was inverted, exhibiting maximal fluorescent intensity at pH 5.0 (Fig 7B). Therefore, low pH is responsible for the disaggregation of MC540 and the increase in fluorescence. It should be noted that incubation with either liposomes or Triton also induced a 10-nm red shift in the MC540 spectrum, from 575 nm to 585 nm.



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Figure 7. Fluorescence spectral analysis of MC540 incubated with either liposomes or Triton. (A) MC540 alone (1,2) or in the presence of 15 µg/ml (3,4) and 30 µg/ml liposomes (5,6) at pH 7.2 and pH 5.0. (B) MC540 alone (1,2) or in the presence of Triton X-100 (3,4) at pH 7.2 and pH 5.0.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

In this study, the subcellular localization of MC540 was investigated in colon carcinoma cells using spectral imaging. For this purpose we chose two cell lines: murine CT26 and human HT29 colon carcinoma cells. Excitation ranges were set to 420–480 nm and 545–580 nm based on the light absorbance properties of MC540. In an aqueous environment MC540 absorbs light near 480 nm, and in lipophilic media and membranes at 570 nm (Ehrenberg and Pevzner 1993 ). However, although the absorbance peak at 570 nm is stronger than at 480 nm, the subcellular distribution pattern of MC540 at both excitation ranges appeared similar. Therefore, the intracellular binding sites of MC540 have both hydrophobic and hydrophilic properties. MC540 is usually considered a membrane probe that binds to the bilayer surface without penetrating deeper than the glycerol level (Anderson et al. 1993 ; Arias et al. 1994 ; Langner and Hui 1999 ), whereas the present spectral imaging results showed localization in cytoplasmic vesicles. An elevated level of MC540 in the cytoplasm has previously been reported in differentiated keratinocytes, but in proliferating cells only the plasma membrane was stained (Giachetti and Chin 1996 ).

In contrast to normal tissues, in which the pH of the extracellular fluid is higher than the intracellular pH, the extracellular pH of tumors is lower and may reach a value of 6.8 ± 0.07, a phenomenon known as the Krebs effect (Stubbs et al. 1994 ). To create an environment similar to that of the tumor, we reduced the pH in the cell culture to 6.7. It is well known that the binding of MC540 to membranes and its fluorescence intensity are influenced by the membrane potential (Fiedorowicz et al. 1993 ) and that a low pH increases membrane potential and the subsequent binding of MC540, resulting in a high fluorescence. All our measurements confirmed that fluorescence of MC540 in the cells increased with the reduction in the pH.

Because MC540 is an anionic dye, repelling forces eliminate its entrance into the negatively charged cell nucleus. However, the reduction in environmental pH enabled its binding to the nuclear membrane in both CT26 and HT29 cells. Furthermore, in proliferating cells MC540 entered the nucleus and associated with the nucleoli. The nucleolar fluorescence could clearly be observed by excitation in the range of 420–480 nm. Therefore, the nucleolar fluorescence is based mainly on hydrophilic binding sites of MC540.

Under acidic conditions we also detected a population of undifferentiated CT26 cells displaying high fluorescence intensity in the plasma membrane. This phenomenon may result from the stress on the lipid bilayer induced by the acidic environment. Changes caused by this stress, such as decrease in cholesterol content, might alter the membrane morphology and increase ion permeability and membrane fluidity (Gaffney et al. 1991 ; Langner and Hui 1999 ). MC540 is able to sense the degree of lipid packing and to insert preferentially into bilayers whose lipids are more widely spaced (Williamson et al. 1983 ).

Although the fluorescence of the MC540 dye was higher at pH 7.2 than at pH 5.0, the fluorescence of MC540-stained cells was maximal at pH 5.0. Incubation in vitro of the MC540 dye with liposomes ruled out the possibility that the increased fluorescence at pH 5.0 is a result of increased binding to membranes. However, incubation of the MC540 dye with Triton X-100 to disaggregate the dye resulted in maximal fluorescence intensity at pH 5.0. Hence, the high fluorescence intensity in acidic conditions can be considered a consequence of an increased binding of MC540 monomers to the cells. In addition, the incubation of MC540 in the presence of either liposomes or Triton X-100 induced a red spectral shift from 575 nm to 585 nm. A red spectral shift has also been observed in hydrophobic domains of MC540-stained cells. Whereas maximal fluorescence intensity in hydrophilic domains was at 580 nm, in hydrophobic areas the fluorescence peak was at 600 nm. This red shift has already been described by Cohen et al. 1974 as it pertains to axons.

The distribution pattern of MC540 was also studied in differentiated CT26 and HT29 cells. Cells treated with sodium butyrate to induce differentiation exhibited cell cycle arrest at G1-phase and an elevated level of ALP. Here, too, MC540 fluorescence was detected solely in cytoplasmic vesicles and not in the plasma membrane. However, cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The same results were obtained in M1 murine myeloid leukemia cells treated with leukemic inhibitory factor to induce differentiation (Chen et al. 1997 ). These differentiation-associated changes in MC540 fluorescence serve as the basis for the discrimination of normal cells from leukemic bone marrow (Kubo and Sieber 1997 ; Pyatt et al. 1999 ).

We can conclude that although MC540 is considered a membrane probe, in colon carcinoma cells its binding is not exclusive to the plasma membrane and may be detected in cytoplasmic vesicles and inside the nucleus. The decrease in MC540 fluorescence with cell differentiation in an environment similar to that of the tumor reflects a change in the binding affinity of MC540 and can be used to distinguish between proliferating and differentiating cells.


  Acknowledgments

We gratefully thank Ms Hana Weitman for technical support and Ms Judith Hanania for help in editing the manuscript.

Received for publication June 14, 2000; accepted August 10, 2000.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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