Fluorochrome Choices for Flow Cytometry

Multicolor flow cytometry requires thorough knowledge not only of your proteins of interest, but also of the corresponding fluorochromes that will be used in your panel. Not all fluorochromes are bright, some are prone to photobleaching, and others are sensitive to fixation and permeabilization. In addition, some proteins are high-density such as lineage markers, whereas others are low-density such as markers associated with differentiation. Multicolor flow cytometry will require compensation, thereby dimming some of your proteins of interest due to corrections in spectral overlap. Use of panel builders and spectrum viewers is instrumental in building panels that will perform optimally. In this chapter, we will review the various fluorochromes available for purchase, highlighting their pros and cons. We will also go over how to use spectrum viewers to estimate the degree of spectral overlap between fluorochromes, and how to optimally pair high- and low-density proteins with certain fluorochromes.