VenturiOne Flow Cytometry Data Analysis Software & Running a Cell Cycle Analysis

Introduction to cell cycle analysis

This article showcases the VenturiOne cell cycle analysis feature in a description of the application of the Watson modelling, in order to overcome the overlap between S phase, G1 and G2/M phases of the cell cycle.

The DNA content of cells varies in relation to the distinct phase of the cell cycle they are currently in. Flow cytometry analyses the different stages of the cell cycle by staining them with a DNA-binding fluorescent dye, such as propidium iodide (Figure 1).

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Figure 1. The relationship between the DNA histogram and the cell cycle.

Frequently, an investigator wants to know the percentage of cells in each phase of the cell cycle particularly in the field of cancer research. Here is where we can observe the effects of cytotoxic drug, radiation and other cancer treatments.

During DNA analysis a problem can arise due to the overlap of cells in G1 and those in early S phase and the overlap between G2/M and late S phase (Figure 2).

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Figure 2. The overlap between S phase, G1 and G2/M phases of the cell cycle. The cells in S phase were labelled with bromodeoxyuridine, which separated the DNA histograms of G1, S and G2/M phases. They then superimposed the three histograms.

To overcome this problem a computer program can separate the phases of the cell cycle by modelling the DNA histogram, and these programs can use several different modelling approaches. One such approach is the modified Watson ‘pragmatic’ algorithm which has been employed in the VenturiOne cell cycle analysis feature.

Experimental procedures

The cells shown in Figure 3 were found to be fixed in 70% ice-cold ethanol, in which they were stored at -20°C where they then, for analysis, were resuspended in PBS with propidium iodide (PI) at 20µg/ml and RNase at 1 mg/ml. For this and other staining protocols see Ormerod, 2000, and Current Protocols in Cytometry, 2007.

Analysis using VenturiOne

The model assumes that G1 and G2/M are normally distributed and fits Gaussian curves to them, returning the peak channel number and the coefficient of variation (CV). It then uses these values to construct the regions of S phase which overlap either with G1 or G2/M, using curves based on the cumulative error function (erf). The model uses the unmodified experimental values for the centre of S phase. Ormerod et al., 1987 describes this in more detail.

When we activate the cell cycle option in the VenturiOne software, a modified Watson algorithm of the cell cycle analysis is automatically applied to the selected histogram plot. The software distinguishes the three phases associated with the DNA cell cycle by colour. Red denotes the G0/G1 phase, blue denotes the S phase and green denotes the G2/M phase.

There are also user adjustable markers present on the cell cycle plots. We have positioned a red marker at the estimated central point of the G0/G1 phase and a green marker at the estimated central point of the G2/M phase. The software displays additional blue markers on the plots to set noise thresholds. It presents events that occur above or below these thresholds on the plots, but does not analyse them.

Some results using the VenturiOne software are shown below. In Figure 3A, a large proportion of cells are in G0/G1 phase as indicated by a large red peak whereas in Figure 3B, the majority of cells are in G2/M phase indicated by a large green peak.

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Figure 3. Cell cycle analysis of (A) a murine leukaemic cell line, (B) the cells 48 hours after incubation with a cytotoxic drug.

Discussion of the Watson model in VenturiOne

Using any program, the goodness of fit will depend on the quality of the DNA histogram. As in the results above, the cell cycle analysis reports an error message which indicates whether the algorithm experienced any problems. Here the message is Fit OK displaying good G1 and G2 CV’s. CV’s across the G1 and G2/M peaks should be =8% should be rejected.

There will be occasions when the program clearly has not fitted the histogram correctly, and the most likely source of the problem is fitting G2 and the S/G2 overlap due to a lot of noise. This is easily detected as the CV G1 and G2/M peaks are vastly out of line, and when this happens you should experiment by changing the starting position of the G2 peak.

The Watson model used in the VenturiOne software is a simple and robust method as it requires the assumption that the data are normally distributed and a recognisable G1 peak; this may be vestigial although from referenced literature, rarely absent. Based on these assumptions, VenturiOne produces fast and simple cell cycle analysis automatically therefore reducing the need for user intervention. As seen in the results, VenturiOne is also used to show differences between typical cell lines and those treated with cytotoxic drugs without having to take anything else into account.

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Note

This program is for research use only.

References

Current Protocols in Cytometry, 2007. John Wiley & Sons, Inc.

Ormerod, M.G. (2000). Flow Cytometry. A Practical Approach.

Ormerod, M.G. (ed). 3rd edition. Oxford University Press, Oxford. 83-97.

Ormerod, M.G., Payne, A.W.R. and Watson, J.V. (1987) Improved program for the analysis of DNA histograms. Cytometry 8: 637-641.

Watson, J.V., Chambers, S.H. and Smith, P.J. (1987) A pragmatic approach to the analysis of DNA histograms with a definable G1 peak. Cytometry 8: 1-8.

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