*The 30 Second Flow Cytometry Lecture

Hematopoietic cells meet the first requirement for analysis by flow cytometry, i.e., cells must be in a single cell suspension. Solid tissues must be disaggregated into single cell components prior to analysis, a limitation that significantly reduces the applications to cell populations that grow as individual cells rather than as a mass.

The dispersed cell population is incubated with antibodies that very specifically bind to cell surface proteins (gene products). Each specific antibody is labeled with a different fluorescent dye, usually green, orange, red or far red, so that cells expressing the particular antigen can be identified by the color of the fluorescence when they pass through an exciting laser beam:

The labeled cells are forced under pressure through a focused laser beam and the colors of fluorescence that are emitted by each cell are quantified by multiple detectors, each measuring a different portion of the light spectrum related to the dyes attached to the antibodies. As a cell passes through the laser beam, morphologic information can also be determined from the cells based on the scattering of light. Light scattered in the forward direction, along the axis of the laser beam, increases with cell size. This is a relative size measurement since the amount of detected light is dependent on the angle of the detector relative to the laser beam, the diameter of the cell and the refractive index of the cellular componentsref. Light scattered at right angles to the laser beam is a result of included granules in the cell, thereby measuring granularity [ref].

As each cell passes the laser beam, signals from each detector are measured and recorded in a computer list associated with that particular cell. As the next cell passes the laser beam, another set of characteristics are recorded for that cell. This list of multiple cell characteristics for each cell in the sample is then saved for further analysis, hence the name “list mode analysis”. The long list of cells, 1,000-1,000,000 events or more, can then be analyzed to detect cells with similar characteristics using specialized programs allowing visualization of the multiple parameters.