Selecting Fluorochrome Conjugates for Maximum Sensitivity

Most users of flow cytometry are aware that, to maximize sensitivity for an antibody whose staining is dim, a bright fluorochrome such as phycoerythrin (PE) should be selected for that conjugate. What is often less well appreciated is the role that spectral overlap plays in determining the sensitivity of a given fluorescence channel. This has become more of an issue with the advent of polychromatic flow cytometry (up to 12 colors) (1) and with the introduction of digital electronics and postacquisition compensation, which can affect resolution, as described below. In this article, the pitfalls of selecting suboptimal conjugate combinations are highlighted, in addition to general rules that should guide conjugate selection. We start from first principles to describe how spectral overlap contributes to the observed background in a given fluorescence channel.