Lesson 5: Immunofluorescence

When people think of flow cytometry, they are generally thinking of an immunofluorescence technique. The binding of fluorescent molecules to cell membranes via antibody conjugation is the most widely used technique in cytometry today.

Cluster Designation

Before discussing antibody conjugation, it is necessary to discuss the general naming of antibodies. In days previous, individual researchers would identify and report on what they thought were novel cell surface antigens. As more an more antigens were discovered and reported on, however, it began to become apparent that many researchers, working independently, were reporting on the same novel antigens. In order to reduce confusion and replication of effort, an international body was formed and to organize antibody labeling, resulting in the Workshops on International Human Leukocyte Differentiation Antigens. (Similar workshops are held for other species-specific antigen labeling)

Via these workshops the antigen cluster designation system was adopted. Each identified antigen is given an official designation, or CD number. A simple cluster designation table can be seen here. As new antigens are identified they are given a temporary CDw (working) number until the next workshop is held for official adoption.

Antibody Conjugation

Antibody conjugation is actually a very simple technique. A typical cell membrane presents a variety of surface antigens. The presence of these antigens, or the lack thereof, relates directly to cellular function. For instance, monocytes are known to present the CD14 antigen on their membrane. Fluorochromes, coupled with an anti-CD14 antibody, can be attached to the cell via this antigen/antibody bond for easy detection. This is known, simply, as direct staining.

The image to the left shows an indirect staining method. In this case, the antibody which attaches to the cellular antigens does not contain a fluorochrome. After staining with this initial antibody, a secondary antibody or other fluorochrome-bearing molecule is introduced which binds specifically to the primary antibody. A popular indirect combination is the biotin/streptavidin pair. In this methodology, one takes advantage of the strong affinity between these two molecules by utilizing a biotinylated primary antibody, and a fluorochrome-labelled streptavidin molecule as a secondary.


Not much need be said at this point regarding fluorochromes. For those that would like a brief reminder on the physics behind fluorescent molecules, Molecular Expressions provides a useful tutorial. (Please bare in mind that this this tutorial relates to applications in microscopy. Therefore, discussions of filter methodology do not entirely apply to the world of flow cytometry).

For our purposes, it is necessary to know two characteristics of a fluorochrome. The excitation and emission spectra. The excitation spectra is necessary to know in order to determine what laser is required to excite the fluorochrome. Many manufacturers will list a fluorochromes peak excitation wavelength. While this lists the wavelength of maximum photon absorption (which is quite useful for microscopists), it is somewhat deceptive as the laser sources used on a typical cytometer have a very high power density, and do not require 100% absorption to provide a good fluorescent response. Knowledge of the emission spectra is, obviously, necessary in order to determine the appropriate detector/filter combinations and to assure minimal spectral overlap in multi-color experiments.

A table of popular fluorochromes may be found here. BDBiosciences provides a java applet which is very useful when determining which fluorochromes to use. When planning experiments, be aware that the FACSCalibur is a two laser system (488nm and 635nm).

Wrapping Up

In order to asses your understanding of the material thus far, please email the answers to the following exercise to the address below. After receiving this I will provide you with access to the next module.

You are asked to decide on a three-color T and B cell enumeration technique. In your email, include the following information:

  • Three antibodies chosen
  • Three fluorochromes chosen
  • The laser(s) wavelengths necessary to excite the chosen fluorochromes
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