Tutorials for building multicolor flow cytometry panels always highlight the importance of antigen density - But why is it so important?
The number of antigens, or target molecules that a cell carries directly correlates to the intensity of the positive population and will determine the optimal fluorophore you should use for each marker. As a general rule you should pair bright fluorophores (e.g. PE) with low expressing markers and dimmer fluorophores (e.g. Pacific Blue) with highly expressed markers. Careful choice of fluorophores will help with the resolution of your cell populations. It is possible for bright fluorophores to be paired with highly expressed antigens, but better to avoid pairing dim fluorophores with low abundance antigens.
Care should be taken however, as fluorophore brightness can have both positive and negative effects.
A bright fluorophore will give better resolution, but have greater spread when compensated, and if placed on an abundant antigen, the increased spillover into adjacent channels may mask the signal from dimmer fluorophores.
Build multicolor flow cytometry panels in just a few simple steps
Fluorophore choice is relatively easy when only a few markers are required. However it becomes more complicated as you increase the number of fluorophores in your panels, especially if the antigen density is unknown or may change with cell subsets or activation state. Knowing the antigen density will help rank markers and pair them to similarly ranked fluorophores. The following guide provides advice on how to measure antigen density and lists the antigen density of some common markers.
If you know the antigen density for any given marker, you can input it in Bio-Rad’s panel builder tool, which will then automatically exclude sub-optimal fluorophores from your panel.
Antigen density can also be useful when determining the biological activity of ligands. The density of receptors has been shown to be related to the biological activity of ligands, e.g. cytokines (Gudipaty et al. 2001; Moraga et al. 2009; Reynes et al. 2000) and their subsequent efficacy.