Principles of Flow Cytometry and Cell Sorting
Revealing the Identity, Function and History of Biological Cells
Francis L. Battye
The Walter & Eliza Hall Institute of Medical research
Flow cytometry (FCM) experiments are becoming increasingly ambitious with attempts at specifying an expanded array of cellular properties. With this increased experimental sophistication comes a concomitant increase in the complexity of instrument operation.
Exquisite cell identification by immunofluorescence has been enhanced by the burgeoning collection of immunofluorochromes, led by the tandem conjugates and now joined by the quantum dots. The range of fluorescent markers for surface phenotyping, which has been the most common FCM measurement for many years, is now challenged by the diversity of fluorescent genetic markers, particularly the plethora of fluorescent proteins.
However it is identified, the current state of a cell (quiescent, activated, proliferating, apoptosing) can also be simultaneously determined. Simple nucleic acid determination can indicate cycling, intra-cellular Ca++ determination can indicate activation and there are many markers of stages of the process of apoptosis.
While a FCM measurement on a cell is instantaneous, it is also possible to glean information about a cell’s history: by the use of a marker for DNA incorporation, whether it was cycling at some time before the measurement or, by using tracking dyes like CFSE, where it came from, or whether it has undergone cell division.
While an increasing proportion of FCM experimenters are no longer trained cytometry specialists, the increased “user-friendliness” of modern instruments allows users to acquire valid data provided they have the required level of technical knowledge of the process. Some of the components requiring prior study are:
- Excitation of fluorescence: Matching available lasers to fluorochromes.
- Detection of fluorescence: Optical filter selection, setting detector sensitivity.
- Signal processing: If the cytometer is largely analog or digital, how does this impinge on the set-up?
- Fluorescence compensation: How does multi-colour compensation differ from pair wise? What can compensation not do for you?
- Gating: When and how should data be gated to remove debris, cell doublets or clumps, and dead cells?