assay protocol

Flow Cytometry Diagram

1. Histogram
Single-parameter histograms display a single measurement parameter (relative fluorescence or light scatter intensity) on the x-axis and the number of events (cell count) on the y-axis.

The histogram shows the total number of cells in a sample that possess certain physical properties selected for or which express the marker of interest. Cells with the desired characteristics are known as the positive dataset. Ideally, flow cytometry will produce a single distinct peak that can be interpreted as the positive dataset. However, in many situations, flow analysis is performed on a mixed population of cells resulting in several peaks on the histogram. In order to identify the positive dataset, flow cytometry should be repeated in the presence of an appropriate negative isotype control. (e.g. if you are using mouse anti-human BID conjugated with FITC as the antibody, then use mouse IgG1 conjugated with FITC as isotype control)

  • Two-parameter histograms display two measurement parameters, one on the x-axis and one on the y-axis, and the cell count as a density (dot) plot or contour map. The parameters could be SSC, FSC or fluorescence.

2. Density blot

An important principle of flow cytometry data analysis is to selectively visualize the cells of interest while eliminating results from unwanted particles e.g. dead cells and debris. This procedure is called gating.

On the density plot, each dot or point represents an individual cell that has passed through the instrument. Lysed whole blood cell analysis is the most common application of gating, and depicts typical graphs for SSC (Side Scatter) versus FSC (Forward Scatter) when using large cell numbers. Normally, the signal of FSC (intensity of FSC light) is closely related to the size of single cell. This means for the same kind of cell, cells with bigger areas brings stronger FSC signals. SSC assay sorts by properties of intracellular organelles and particles. Though it's affected by shape and size of cells, it's more sensitive to membranes, cytoplasm, nucleus etc. The different physical properties of granulocytes, monocytes and lymphocytes allow them to be distinguished from each other and from cellular contaminants.

3. Contour diagram

Contour diagrams are an alternative way to demonstrate the same data as density plot does. Joined lines represent similar numbers of cells. The graph takes on the appearance of a geographical survey map, which, in principle, closely resembles the density plot. It is a matter of preference but sometimes discreet populations of cells are easier to visualize on contour diagrams.