Flow Cytometry (FACS) Specificity and Non-Specificity

In order to draw conclusions about particular proteins on cells, we have to be sure that the stains that we use are specific in their binding to the proteins in which we are interested. Lack of specificity may occur as a result of several factors: Firstly, an antibody may bind to a common epitope on several different proteins. Secondly, different sorts of cells contain the same proteins, so a staining reaction may not be as specific as one would like in classifying different cell types. These first two types of problems require knowledge of the appropriate reagents for answering particular questions. Two other types of specificity problems are more difficult to control. Antibodies bind to many cell types by their non-specific (Fc) ends. Monocytes, in particular, professionally bind many antibodies through their Fc-receptors. The use of Fab or F(ab')2 fragments (antibodies without their Fc ends) is recommended whenever possible and helps to alleviate this problem. Blocking the Fc-receptors (see below) also helps. Finally, dead cells, with compromised membrane integrity, tend to be sticky and to bind all sorts of reagents with careless abandon. The dead cell problem can be helped by careful cell preparation or by "gating out" dead cells from analysis -- either through propidium iodide staining to positively identify dead cells by their membrane permeability or, less securely, through avoiding cells with low FSC intensity (cells that are dead usually show low FSC signals probably because the refractive index of their cytoplasm is similar to that of the surrounding medium). In the final analysis, however, in order to permit conclusions from fluorescence staining about the classification or chemical constituents of a cell, flow experiments must make use of careful controls.