Flow book denovo software

If you have downloaded the free FCS Express Reader , you can open the analysis layout that was used to create each figure. In some cases, extra information about the figure is supplied, including other data files. The reader can experiment with the data, changing the position of the gates, markers, quadrants, etc. To get an overview of the book and the intended audience, please read the Preface.

All rights reserved. I will, of course, acknowledge any contribution. I would like to thank the many colleagues who have worked with me over the years and, in particular, Mrs. Jenny Titley, who recorded much of the data shown in the handbook. The contributions of other colleagues who have been so generous in supplying me with data are acknowledged in the text. I would also like to thank Drs. Michael G. May, Ormerod Preface Flow cytometry is widely used in biological research.

Ormerod m. This plot can be used to set a region for the light scatter gate for the epithelial cells. It will include some granulocytes but will have excluded the bulk of the leukocytes. Human carcinoma cells plus peripheral blood. Scatter plot note that the side scatter is logarithmic. Some of the larger epithelial cells are off-scale on the FS axis. With hindsight, the FS should have been measured on a log scale.

For details, see Figure 4. This region has then been used to gate on the light scatter plot C. The leucocyte subsets have also been identified on the basis of their side scatter and lack of expression of Ber-EP4. Two measures are generally made of a distribution, intensity and spread. If the data has been displayed on a linear scale, the arithmetic mean is used; for logarithmically displayed data, the geometric mean is generally chosen.

If any part of the distribution lies off scale at either end of the axis, the value for the mean channel number will be inaccurate and should not be used; the median channel can be used as long as more than half of the distribution in on scale. The peak channel number is an inaccurate measure of the centre of a distribution and is not recommended. For a Guassian normal distribution, these three values should be equal.

The spread of a distribution is usually expressed as the Standard Deviation SD. However, in flow cytometry, the coefficient of variation CV is preferred because it is dimensionless and, on a linear scale, does not depend on where in the histogram the data is recorded. Frequently the percentage of cells in a sub-population is required. In immunofluorescence analysis, quadrants are often drawn on a cytogram and the number of cells in each quadrant recorded.

While quadrants are often convenient to use, they are not always required Figure 4. Quadrant regions showing the percentage of cells in each sub-population. Neuroblastoma cells incubated with BrdUrd and labelled with an anti-BrdUrd antibody and propidium iodide, which binds to DNA and shows the cell cycle. For further information about this method, see Chapter 8. While flow cytometry generally gives the percentage of a particular sub-set of cells, some flow cytometers precisely record the the volume of sample analysed or deliver a fixed volume of sample.

A percentage count of a sub-population of cells can be directly converted to an absolute count. In instruments without this facility, two approaches are used to measure the absolute count, referred to as two platform or one platform methods.

In the two platform approach, the concentration of all the cells in a sample is determined by another method. For blood leucocytes, a haematology counter is used. For cultured cells, an electronic sizing Coulter counter may be used. The flow cytometer is then used to determine the percentage of cells in a particular sub-set so that the cell concentration of the sub-set can be calculated.

The disadvantage of the two platform method is that errors in the two instruments are compounded and that two instruments are required. In the one platform method, an absolute cell count is performed on the cytometer. Some instruments allow a fixed volume of sample to be analysed, which will give an absolute count directly.

In most cytometers, a fixed volume of sample is spiked with a known number of fluorescent beads. Suitable beads with an assayed concentration may be obtained from Beckman Coulter; alternatively, tubes containing known numbers of lyophilised beads are sold by BD.

The brightness and light scatter of the beads is different to that of cells so that beads and cells can be easily distinguished in the flow cytometer. Counting the number of beads in the portion of the sample analysed allows the volume analysed to be calculated and hence the concentration of cells.

The disadvantage of this method is that beads can stick to each other and to the walls of the tube leading to an underestimation of the bead count. When recording the percentage of positive cells, we need to distinguish between negative and positive staining, that is, to define the negative population.

To define the negative cells, unstained cells are inadequate; they will only correct for autofluorescence and ignore any fluorescence resulting from non-specific binding to the cells. If the sample contains negative cells as, for example, in Figure 4. If the samples does not contain negative cells, people have traditionally used an isotype control.

For example, if cells are stained with a fluorescein-labelled mouse IGg1 antibody, labelled IgG1 fraction is used as the control.