Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry

BACKGROUND: Caspase activation is a critical early step in the onset of apoptosis. Cell-permeable fluorogenic caspase substrates have proven valuable in detecting caspase activation by flow cytometry. Nevertheless, detection of early low-level caspase activation has been difficult using conventional area or peak fluorescence analysis by flow cytometry, despite the apparent presence of these cells as observed by microscopy. We describe a method utilizing maximum fluorescence pixel analysis by laser scanning cytometry (LSC) to detect early apoptotic cells. METHODS: The PhiPhiLux-G(1)D(2) caspase 3/7 substrate was used in combination with DNA dye exclusion and annexin V binding to identify several stages of apoptosis in EL4 murine thymoma cells by both traditional flow and LSC. LSC analysis of maximum pixel brightness in individual cells demonstrated an intermediate caspase-low subpopulation not detectable by flow or LSC integral analysis. LSC analysis of caspase activity was then carried out using the larger UMR-106 rat osteosarcoma cell line to determine if this apparent early caspase activity could be correlated with localized, punctate caspase activity observed by microscopy. RESULTS: The caspase-low subpopulation found in apoptotic EL4 cells was also observable in UMR-106 cells. Relocation to cells with low fluorescence due to caspase activity and subsequent examination by microscopy demonstrated that these latter cells indeed show punctate, highly localized caspase activation foci that might represent an early stage in caspase activation. CONCLUSIONS: Cells with low-level, localized caspase expression can be detected using maximum pixel analysis by LSC. This methodology allows an early step of apoptotic activation to be resolved for further analysis.     

Measurement of DNA damage associated with apoptosis by laser scanning cytometry.

BACKGROUND: Phosphatidylserine (PS) binding by annexin V (AV) is an early membrane marker of apoptosis. Using laser scanning cytometry (LSC) and the comet assay, we showed that the DNA of AV(+) cells is so highly fragmented that it cannot be quantified by the comet assay (Bacso et al.: Cancer Res 60:4623-8, 2000). METHODS: The "halo" assay was used instead of the comet assay to quantify DNA damage associated with apoptosis. The LSC was used to measure both AV fluorescence and DNA damage on the same Jurkat cells following treatment with anti-Fas. The data from both sets of measurements were merged, allowing direct correlation of membrane and nuclear markers of cell death. RESULTS: AV(+) cells had significant DNA damage determined by the ratio between nuclear DNA and peripheral (migrated) DNA. Cells in the early and late stages of apoptosis could be discriminated on the basis of DNA content. In addition, it was possible to distinguish between apoptotic and necrotic cells in the AV(+) propidium iodide-positive population based on DNA content and DNA damage. The addition of specific inhibitors for caspases-8, 9, and 3 blocked both PS externalization and DNA fragmentation, indicating these events are downstream from caspase activation. CONCLUSIONS: This technique allows accurate distinction between apoptotic and necrotic cells and cytometric grading of apoptosis.     

Laser scanning cytometry in the characterization of the proapoptotic effects of transiently transfected genes in cerebellar granule neurons.

BACKGROUND: Low transient transfection efficiency limits the ability to characterize putative proapoptotic gene function in neurons. Laser scanning cytometry (LSC), with its high capacity, medium throughput means of collecting fluorescent emissions from cultured cells, offers an effective technology for scoring cell death in neuronal transfectants. METHODS: Cerebellar granule neurons (CGNs) were transfected with EGFP-fusion constructs of Caspase-3 and Caspase-9 using a DNA-calcium phosphate coprecipitation method. CGNs were fixed, permeablized, and stained with propidium iodide (PI) nuclear dye. An LSC method, based on a combination of Long Red Max Pixel, Long Red Integral, and Green Integral fluorescence parameters was validated for the scoring of apoptotic cell death in CGNs. RESULTS: In Caspase-3 and Caspase-9 transfected CGNs, cell death was scored both in transfectants and nontransfected culture-mates. The cell death phenotype was found to be independent of transfection efficiency. LSC scoring of Caspase-9 transfectants was compared with visual scoring following Hoechst 33342 staining, yielding results that were similar qualitatively, but not quantitatively, likely owing to the greater sensitivity to green fluorescence of laser scanning compared to human vision. CONCLUSION: LSC scoring of transiently transfected CGNs offers a rapid and reliable means of characterizing proapoptotic gene effects.     

Translocation of Bax to mitochondria during apoptosis measured by laser scanning cytometry

BACKGROUND: During induction of apoptosis, the pro-apoptotic member of the Bcl-2 protein family (Bax) undergoes translocation to the mitochondria. The translocation, which leads to accumulation of Bax in the mitochondrial intermembrane space, appears to be the critical event determining release of cytochrome c to cytosol: the latter event triggers the irreversible steps of apoptosis, namely, the activation of caspases and the initiation of the degradation of many proteins. The aim of this study was to utilize the morphometric capabilities of the laser scanning cytometer (LSC) and adapt this instrument to detect and measure in situ the process of translocation of Bax to mitochondria. METHODS: Human breast carcinoma MCF-7 cells growing on microscope slides were treated with the DNA topoisomerase I inhibitor, camptothecin (CPT). CPT is known to induce apoptosis preferentially of S-phase cells. The cells were fixed and permeabilized on the slides, their DNA was stained with propidium iodide (PI), Bax was detected immunocytochemically with the fluoresceinated antibody, and red and green fluorescence emission was measured by the LSC. RESULTS: Prior to induction of apoptosis, Bax was uniformly and diffusely dispersed in the cell nucleus and cytoplasm. Its translocation and accumulation in mitochondria in cells undergoing apoptosis were detected and measured by the LSC as the increase in intensity of maximal pixel of Bax immunofluorescence. Bivariate analysis of DNA content versus maximal pixel of Bax fluorescence revealed that the CPT-induced Bax translocation into mitochondria was preferential to S-phase cells. Total cellular Bax immunofluorescence measured by flow cytometry, however, was increased in all phases of the cycle without a preference to S-phase cells. CONCLUSION: Changes in abundance and localization of particular proteins that undergo translocation within the cell, leading to their altered local density, may be conveniently detected by the LSC by taking advantage of its morphometric capabilities. Measurement of total cellular Bax immunofluorescence by flow cytometry combined with analysis of its translocation by LSC revealed that apoptosis of S-phase cells induced by CPT was unrelated to overall Bax abundance per cell but correlated with its accumulation in mitochondria.     

Exposure of cells to static magnetic field accelerates loss of integrity of plasma membrane during apoptosis

BACKGROUND: Much attention is being paid to the biologic effects of magnetic fields (MFs). Although MFs enhance tumorigenesis, they are neither mutagenic nor tumorigenic. The mechanism of their tumorigenic effect has not been elucidated. METHODS: To investigate the effect of MFs on apoptosis in HL-60 cells, we exposed the cells to static MFs of 6 mT generated by a magnetic disk of known intensity. Apoptosis was triggered by the DNA topoisomerase I inhibitor, camptothecin (CPT). Activation of caspases in situ using the fluorochrome-labeled inhibitor (FLICA) method and determination of plasma membrane integrity by excluding propidium iodide (PI) were measured by both laser scanning cytometry (LSC) and flow cytometry (FC). LSC and FC identified cells at three sequential stages of their demise: early apoptosis (cells with activated caspases and PI negative); late apoptosis (cells with activated caspases but unable to exclude PI); secondary necrosis (cells with apoptotic morphology no longer stained with FLICA, not excluding PI). RESULTS: MF alone did not induce any apoptogenic or necrogenic effect. CPT exposure led to the sequential appearance of apoptotic cells. In the presence of CPT and MF, the overall proportion of cells undergoing apoptosis was not significantly changed. However, we consistently observed a significant increase in the frequency of late apoptotic/necrotic cells when compared with samples treated with CPT alone (P < 0.001), as well as a decrease in the percentage of early apoptotic cells (P = 0.013). The data obtained by FC and LSC were consistent with each other, showing a similar phenomenon. CONCLUSION: Whereas MF alone or with CPT did not affect overall cell viability, it accelerated the rate of cell transition from apoptosis to secondary necrosis after induction of apoptosis by the DNA-damaging agent, CPT. Modulation of the kinetics of the transition from apoptosis to secondary necrosis by MF in vivo may play a role in inflammation and tumorigenesis.     

Prolonged apoptosis in mitochondria-rich cells of tilapia (<Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>) exposed to elevated salinity

The time-course of programmed cell death (apoptosis) during reorganization of gill epithelium in salinity-stressed tilapia was analyzed using a recently developed method based on laser scanning cytometry (LSC) of dissociated gill cells. Apoptosis in mitochondria-rich cells (MRC) was distinguished from that in other cell types using Na⁺/K⁺ ATPase (NKA) as a cell-specific marker. Caspase 3/7 activity in MRC, assessed using LSC and microplate assays, increased significantly starting at 6 h of salinity stress and remained elevated for at least 5 days. This time-course of apoptosis in MRC during acute salinity stress was reflected in elevated apoptotic DNA fragmentation. In parallel to induction of apoptosis, MRC showed a pronounced shift to G2 phase of the cell cycle, which is indicative of G2/M cell cycle arrest, and an increase in NKA abundance per MRC. Unlike in MRC, apoptosis was not significantly increased in other gill cell types, although there was a small transient increase in DNA fragmentation at 6 h. G2 arrest was also observed. Overall, we interpret our data as evidence for a significant role of apoptosis in the extensive reorganization of MRC populations that takes place during salinity acclimation, perhaps similar to its well-established role during organismal development.     

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.     

胡桃楸提取液诱导Hela细胞凋亡的研究

目的：初步探讨胡桃揪提取液对Hela细胞的凋亡诱导作用。方法：通过形态学观察,琼脂糖凝胶电泳及流式细胞术（FCM）检测凋亡细胞。结果：形态学观察发现核固缩、出现凋亡小体。琼脂糖凝胶电泳可见DNA ladder条带。流式细胞仪检测有凋亡峰。结论：胡桃揪提取液可诱导Hela细胞凋亡。     

Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry

The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3',4',5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h.     

Concanavalin A-induced apoptosis in murine macrophages through a Ca(2+)- independent pathway

Concanavalin A (ConA), normally a mitogen of T lymphocytes, was found to induce apoptosis or programmed cell death in murine peritoneal macrophages. The following observations support this assertion: 1) incubation of peritoneal macrophages or cultured PU5-1.8 macrophage cells with ConA caused a dose- and time-dependent reduction of mitochondrial dehy-drogenase activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 2) treatment of cells with ConA induced formation of apoptotic bodies as seen under the confocal laser scanning microscope, 3) challenge of cells with ConA produced a considerable amount of cell debris with DNA content next to G0 phase as revealed by flow cytometry and 4) ConA was able to elicit DNA fragmentation in these cells. The involvement of Ca(2+) in mediating the apoptosis was studied in single cells by confocal laser scanning microscope using the Ca(2+) fluorescence dye, fluo-3. Our results show that ConA induced an immediate rise of intracellular free Ca(2+) concentration as well as opening of Ca(2+) channels on cell surface. But when the cells were treated with 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/AM (BAPTA/AM), a Ca(2+) chelator, to buffer the rise of internal Ca(2+), ConA still caused DNA fragmentation. Furthermore, injection of Ca(2+) into the cell with ionomycin had no stimulatory effect on DNA fragmentation. These results suggest that Ca(2+) changes induced by ConA are not a prerequisite for apoptosis in macrophages.     

Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG-7)

We have demonstrated a method for the in situ determination of the cell cycle phases of TIG-7 fibroblasts using a laser scanning cytometer (LSC) which has not only a function equivalent to flow cytometry (FCM) but also has a capability unique in itself. LSC allows a more detailed analysis of the cell cycle in cells stained with propidium iodide (PI) than FCM. With LSC it is possible to discriminate between mitotic cells and G₂ cells, between post-mitotic cells and G₁ cells, and between quiescent cells and cycling cells in a PI fluorescence peak (chromatin condensation) vs. fluorescence value (DNA content) cytogram for cells stained with PI. These were amply confirmed by experiments using colcemid and adriamycin. We were able to identify at least six cell subpopulations for PI stained cells using LSC; namely G₁, S, G₂, M, postmitotic and quiescent cell populations. LSC analysis facilitates the monitoring of effects of drugs on the cell cycle.     

Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry

Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.     

Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

Flow cytometry and factor analysis evaluation of confocal image sequences of morphologic and functional changes occurring at the mitochondrial level during 7-ketocholesterol-induced cell death

OBJECTIVE: To analyze functional and morphologic alterations that occur at the mitochondrial level by flow cytometry and laser scanning confocal microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Under treatment of U937 cells with 7-ketocholesterol, functional alterations that occur at the mitochondrial level (especially loss of transmembrane mitochondrial potential [delta psi m]) were assessed with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and mitotracker red (CMXRos), whereas morphologic changes were analyzed with nonyl acridine orange (NAO). By flow cytometry, these different dyes were excited at 488 nm, whereas on CLSM, excitation of NAO and CMXRos was performed by lines of an argon laser. By CLSM, spectral sequences were performed to characterize NAO and CMXRos. FAMIS was used to transform the image sequences in factor images. RESULTS: By flow cytometry, rapid loss of delta psi m induced by 7-ketocholesterol was detected with both DiOC6(3) and CMXRos, which gave similar results. Morphologic alterations of mitochondria were revealed with NAO. The factor images obtained from confocal image sequences confirmed these results. CONCLUSION: The simultaneous use of NAO, CMXRos and FAMIS constitutes a new method to detect morphologic and functional alterations occurring at the mitochondrial level during cell death.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Intranuclear localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma

OBJECTIVE: To investigate, with laser scanning cytometry (LSC), proliferating cell nuclear antigen (PCNA) expression during the cell cycle in renal cell carcinoma. STUDY DESIGN: DNA ploidy and intracellular localization of PCNA in renal cell carcinoma were determined using LSC and immunohistochemistry. The subjects were nine patients who had received surgery for renal cell carcinoma. After DNA ploidy analysis, the glass slides were restained by immunohistochemistry of PCNA. LSC allowed direct observation of PCNA localization during the cell cycle because we could obtain immunohistochemical staining of PCNA as a function of cell cycle phase for individual cells. RESULTS: PCNA was not demonstrated in the nuclei of G0/G1 cells. PCNA expression increased from the S phase of the cell cycle. PCNA rapidly degraded at the end of the G2 phase. In the late G2 and M phase, PCNA was not detected in almost any nucleus. CONCLUSION: LSC allows morphologic observation of the intracellular distribution of PCNA during the cell cycle in renal cell carcinoma.     

Inference of cell cycle-dependent proteolysis by laser scanning cytometry

Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is employed, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods.     

Multiparameter analysis of human epithelial tumor cell lines by laser scanning cytometry

Laser scanning cytometry (LSC) is a relatively new slide-based technology developed for commercial use by CompuCyte (Cambridge, MA) for performing multiple fluorescence measurements on individual cells. Because techniques developed for performing four or more measurements on individual lymphoid cells based on light scatter as a triggering parameter for cell identification are not suitable for the identification of fixed epithelial tumor cells, an alternative approach is required for the analysis of such cells by LSC. Methods for sample preparation, event triggering, and the performance of multiple LSC measurements on disaggregated fixed human cells were developed using normal lymphocytes and two human breast cancer cell lines, JC-1939 and MCF-7, as test populations. Optimal conditions for individual cell identification by LSC were found to depend on several factors, including deposited cell density (cells per unit area), the dynamic range of probe fluorescence intensities, and intracellular distribution of the fluorescent probe. Sparsely deposited cells exhibited the least cell overlap and the brightest immunofluorescent staining. Major advantages of using DNA probes over a cytoplasmic immunofluorescent protein marker such as tubulin for event triggering are that the former exhibit greater fluorescence intensity within a relatively sharply demarcated nuclear region. The DNA-binding dye LDS-751 was found to be suboptimal for quantitative DNA measurements but useful as a triggering measurement that permits the performance of simultaneous fluorescein isothiocyanate-, phycoerythrin-, and indodicarbocyanine-based measurements on each cell. A major potential advantage of LSC over flow cytometry is the high yields of analyzable cells by LSC, permitting the performance of multiple panels of multicolor measurements on each tumor. In conclusion, we have developed and optimized a technique for performing multiple fluorescence measurements on fixed epithelial cells by LSC based on event triggering using the DNA-binding dye LDS 751. Although not ideal for quantitative measurements of cell DNA content, the large Stokes shift of this dye permits the performance of three or more additional fluorescence measurements on each cell.     

A new approach for the analysis of testicular cells using a laser scanning cytometer.

The laser scanning cytometer (LSC) is a new laboratory tool that offers increased sensitivity and specificity compared to traditional technology. By combining the properties of the advantages of flow cytometry and immunohistochemistry, LSC-based analysis allows the automated evaluation of testicular cells in general and meiosis in particular. Testicular cell smears with previous staining by propidium iodide were analyzed by LSC. The results were compared with those for flow cytometry. LSC is a new, applicable methodology for analyzing spermatogenesis schedule.     

Micronuclei assay by laser scanning cytometry

BACKGROUND: The micronuclei (MN) assay is used to assess the chromosomal/mitotic spindle damage induced by ionizing radiation or mutagenic agents in vivo or in vitro. Because visual scoring of MN is cumbersome semi-automatic procedures that relay either on flow cytometry or image analysis were developed: both offer some advantages but also have shortcomings. METHODS: In the present study laser scanning cytometer (LSC), the instrument that combines analytical capabilities of flow and image cytometry, has been adapted for quantitative analysis of MN. The micronucleation of human breast carcinoma MCF-7 and leukemic HL-60 and U-937 cells was induced by in vitro treatment with mitomycin C. Cellular DNA was stained with propidium iodide (PI), protein was counterstained with fluorescein isothiocyanate (FITC). Two approaches were used to detect MN: (a) the threshold contour was set based on the data from the photosensor measuring red fluorescence of PI and MN were identified on the bivariate PI versus PI/FITC fluorescence distributions by their characteristic position; (b) the threshold contour was set on the data from the sensor measuring FITC fluorescence which made it possible, using the LSC software dedicated for FISH analysis, to assay both the frequency and DNA content of individual MN within each measured cell. RESULTS: The capability of LSC to relocate MN for visual examination was useful to confirm their identification. Visual identification of MN combined with their multiparameter characterization that took into an account their DNA content and protein/DNA ratio made it possible establish the gating parameters that excluded objects that were not MN; 93.3+/-3.3 events within the selected gate were MN. It was also possible to successfully apply FISH software to characterize individual cells with respect to quantity of MN residing in them. The percentage of MN assayed by LSC correlated well with that estimated visually by microscopy, both for MCF-7 (r = 0.93) and HL-60 cells (r = 0.87). CONCLUSIONS: LSC can be used to obtain unbiased estimate of MN frequencies. Unlike flow cytometry, it also allows one to characterize individual cells with respect to frequency and DNA content of MN residing in these cells. These analytical capabilities of LSC may be helpful not only to score MN but also to study mechanisms by which clastogenic agents induce MN.