The cells analysed by LSC have to be deposited either on microscope slides or on multiwell culture plates. In the case of cells adapted to grow attached to culture flasks, the most convenient approach is to maintain them on mono- or multi-chamber microscope slide tissue culture vessels such as provided by the Lab-Tek, Nalge Nunc, Naperville, IL, USA (37
). The subsequent steps of fixation, fluorochrome staining and fluorescence intensity measurement are then carried out with no need for cell detachment (trypsinization), on the same platform on which they were exposed to the agents expected to induce MN in cultures. In the case of cells that grow in suspension, the initial step, prior to fixation, is to deposit them on microscope slides by cytocentrifugation (38
It has been observed that concurrent differential staining of DNA and protein of the cells subjected to MN analysis by LSC with fluorochromes of different emission colour is more advantageous than staining DNA alone. This is due to the fact that the ratiometric analysis of protein/DNA versus DNA content offers better means of MN identification than DNA content alone (38
). A variety of fluorochromes can be used to differentially stain cellular DNA and protein within a given sample. A simple approach, in which fluorescence is excited with a single 488 nm laser, utilises propidium iodide (PI) and fluorescein isothiocyanate (FITC) as DNA and protein fluorochromes, respectively (41
). The use of PI to selectively stain DNA requires removal of RNA which is accomplished by incubation of the fixed and permeabilized cells with RNase A. Alternatively, DNA can be stained with 4′-6-diamidino-2-phenylindole (DAPI), 7-aminoactinomycin D (7-AAD) or other DNA-specific fluorochromes with no need for RNase treatment (42
Two strategies (methods) can be used to measure fluorescence intensity of the protein (e.g. FITC) and DNA-bound (e.g. PI) fluorochromes in assessing frequency of MN by LSC. In the first method (), the ‘threshold’ contour is set based on the data computed from the photomultiplier measuring red fluorescence of PI. The ‘integration’ contour is then set within a range between zero and two pixels outside the threshold contour. In this way, the integral values of DNA (PI)- and protein (FITC)-associated fluorescence intensity of nuclei as well as MN are recorded in the same file. The distinction between nuclei and MN is then made based on difference in their DNA content. The data thus resemble those obtained by FC as the latter also rely on analysis of DNA content alone (20
). As mentioned, however, the concurrent analysis of DNA and protein content of MN, in particular, the ratio of protein/DNA, which is similar in nuclei and MN, provides an additional parameter useful to distinguish MN from artifacts ().
Fig. 2 Two different strategies for setting the threshold contour. (A) The mitomycin C-treated MCF-7 cells were fixed and then stained with FITC and PI. The threshold contour was set on red fluorescence of PI and the integrated values of green (FITC) and red (more ...)
Fig. 3 Identification of MN based on analysis of DNA content (A) and protein/DNA ratio (B). To induce MN, HL-60 cells were treated with mitomycin C, then fixed and stained with FITC and PI. The threshold contour was set on the data from the photomultiplier measuring (more ...)
The second strategy makes use of the feature of LSC software that was designed for fluorescence in situ
hybridisation (FISH) analysis (43
). In this method, the threshold contour is set on the protein-associated (green-FITC) fluorescence (). Each cell is therefore identified, which allows one to obtain information about the number of nuclei and MN per individual cell (number of ‘FISH spots’), as well as to measure intensity (integrated value) of red (DNA) and green (FITC) fluorescence per each spot as well as per whole cell (nucleus + MN). The value of DNA-associated fluorescence integrated over nucleus + MN provides information on the cell cycle position, discriminating between G1
, S and G2
M cells. A similar strategy of threshold contouring based on cellular protein-associated fluorescence has been used to analyse individual cells within cell colonies (44
The capability of LSC to obtain and save images of the measured events allows their visual identification and thus makes it possible to accurately distinguish and separate MN from other objects, primarily cell fragments and debris. The image analysis revealed that >93% of the objects localised within the bivariate distribution window spanning the range between 0.1 and 5% of DNA content (PI fluorescence) of that of nuclei of G1
(diploid) cells and having similar protein/DNA (FITC/PI) ratio as the nuclei () were MN (38
). Thus, on the bivariate PI versus FITC/PI fluorescence plots, three distinct clusters can be seen: (i) the cluster of the recorded events with the highest FITC/PI ratio and the lowest PI fluorescence which are the non-specific particles, mainly fragments of cells’ cytoplasm; (ii) the cluster representing whole nuclei that had the highest PI fluorescence with the typical pattern reflecting the G1
M cell cycle and (iii) the cluster representing predominantly MN.
Analysis of MN using the FISH approach is illustrated in . Setting the threshold contour on FITC fluorescence makes it possible to record each individual cell and count the frequency of the objects emitting PI fluorescence such as MN (‘FISH spots’) and also to measure the integrated PI fluorescence over the whole cell (nucleus + MN). Thus, the cells with a single nucleus could be distinguished from the cells having a nucleus and one MN, from the cells having one nucleus and two MN, etc. It is evident from this data that in the cultures treated with increasing doses of mitomycin C (a cytotoxic and genotoxic drug), the percentage of cells without MN decreased concurrently with the increase in frequency of the cells with one, two and more MN.
Fig. 4 Quantification of MN per cell using the FISH-dedicated software of LSC. The mitomycin C-treated HL-60 cells were fixed and stained with FITC and PI. The threshold contour was set on green fluorescence of FITC, as shown in and the data were collected (more ...)
To make the conditions of analysis of chromosome damage independent of the cell cycle kinetics, the MN assay has to be restricted to cells that made only a single division after exposure to the damaging agent. Towards this end, cytochalasin B is added into cultures to prevent cytokinesis in cells completing nuclear division after genotoxin exposure (1
). In this CBMNcyt assay, only cells that have completed one nuclear division, identified as binucleated cells, are scored. Further nuclear division in the presence of cytochalasin leads to formation of multi-nucleated cells, which are not scored. The strategy of setting the threshold contour on green (FITC) fluorescence combined with selection of cells within a specific range of cellular DNA content can be used to adapt the CBMNcyt assay to LSC (). Specifically, the cytochalasin-arrested binucleated cells are expected to contain DNA content between 2.0 (both nuclei in G1
) and 4.0 DNA index (DI) (both nuclei in G2
). However, the binucleated cells with 2.0 DI overlap on DNA content frequency histograms with single-nucleated G2
-phase cells. Furthermore, the tetra-nucleated cells containing G1
-phase nuclei may have 4.0–8.0 DI DNA content and overlap in DNA content with binucleated cells containing G2
-phase nuclei. Therefore, the range of cellular DNA content between 2.2 and 3.8 DI is the most reliable to represent the binucleated cells. Indeed, imaging of cells whose PI fluorescence (DNA content) was within this range confirmed that >80% of these cells were binucleated (38
). The remaining objects were aggregates consisting of two or three cells in close proximity to each other; the contouring can mistakenly recognise such aggregates as single cells. Strategies that can be used to overcome the problem of close cell proximity or overlap in analysis of MN by LSC are discussed at the end of this chapter: ‘Potential challenges’.
Fig. 5 Identification of cytochalasin B induced binucleated cells by gating analysis of the DNA content frequency histograms. U-937 cells were in the culture with cytochalasin B for 24 h, then fixed and stained with FITC and PI. The threshold contour was set (more ...)
When cultured cells were treated with mitomycin C, the frequency of MN detected visually by microscopy at different mitomycin C concentrations correlated well with that assessed by LSC in both cases evaluated in binucleated cells (). The highest MN frequency (12–14%) was seen at 0.1 μg/ml concentration. However, the MN frequency was diminished at both <0.1 μg/ml and >0.1 μg/ml concentrations of mitomycin C. At 10.0 μg/ml mitomycin C concentration, the cells were arrested in the G2 phase (as was evident from the DNA content frequency histograms) which prevented cells from completing nuclear division and expressing the damage as MN. When the frequency of MN in the same specimens was analysed visually by microscopy and compared with that assayed by LSC, in double-blind tests, rather good correlation (r = 0.87) was observed between both assays (See legends to and ).
Fig. 6 Comparison of frequency of MN binucleated cells in relation to concentration of mitomycin C assessed visually by microscopy (white bars) and by LSC (black bars). MCF-7 cells were treated with different concentrations of mitomycin C for 6 h then transferred (more ...)